Spiro compounds as inhibitors of fibrinogen-dependent platelet aggregation

ABSTRACT

This invention relates to certain spirocyclic compounds substituted with both basic and acidic functionality, as shown by formula (I):wherein Q, L, Ai, Bj, R0, R3, R10, m, n, p and q are as defined in the disclosure, which are useful in inhibiting of platelet aggregation.

This application is a 371 of PCT/US96/15703 filed Sep. 27, 1995 whichclaims the benefit of Provisional Application 60/004,557 filed Sep. 29,1995.

FIELD OF THE INVENTION

This invention relates to novel spiro compounds useful as glycoproteinIIb/IIIa antagonists for the prevention of thrombosis.

BACKGROUND OF THE INVENTION

The most prevalent vascular disease states are related to plateletdependent narrowing of the blood supply such as atherosclerosis andarteriosclerosis, acute myocardial infarction, chronic stable angina,unstable angina, transient ischemic attacks and strokes, peripheralvascular disease, arterial thrombosis, preeclampsia, embolism,restenosis following angioplasty, carotid endarterectomy, anastomosis ofvascular grafts, and etc. These conditions represent a variety ofdisorders thought to be initiated by platelet activation on vesselwalls.

Platelet adhesion and aggregation is believed to be an important part ofthrombus formation. This activity is mediated by a number of plateletadhesive glycoproteins. The binding sites for fibrinogen, fibronectinand other clotting factors have been located on the platelet membraneglycoprotein complex IIb/IIIa. When a platelet is activated by anagonist such as thrombin the GPIIb/IIIa binding site becomes availableto fibrinogen, eventually resulting in platelet aggregation and clotformation.

Heretofore it has been proposed to block these thrombus formation sitesby the use of various therapeutic agents.

There is a need in the area of cardiovascular and cerebrovasculartherapeutics for new agents which can be used in the prevention andtreatment of thrombi.

It is a discovery of this invention that certain novel spiro compoundsblock the GPIIb/IIIa fibrinogen receptor, thereby inhibiting plateletaggregation and subsequent thrombus formation. Pharmaceuticalformulations containing the spiro compounds of this invention inhibitaggregation and are useful for the prophylaxis and treatment ofthrombogenic diseases, such as myocardial infarction, angina, stroke,peripheral arterial disease, disseminated intravascular coagulation andvenous thrombosis.

SUMMARY OF THE INVENTION

The present invention covers novel spiro compounds having a Spironucleus formed from two fused rings, A and B, represented by the formula(I), as hereinafter defined, and all pharmaceutically-acceptable salts,solvates and prodrug derivatives thereof:

having substituents and subscripts; Q, —(L)—, A_(i), p, R₁₀, m, n, R₀,B_(j), q, and R₃, as hereinafter defined.

Another aspect of the invention is a pharmaceutical formulationcontaining a novel spiro compound of the invention.

Another aspect of the invention is a method of inhibiting plateletaggregation, inhibiting fibrinogen binding, or preventing thrombosis byadministering to a mammal the novel Spiro compounds of the invention.

Another aspect of this invention is a method of treating a human toalleviate the pathological effects of atherosclerosis andarteriosclerosis, acute myocardial infarction, chronic stable angina,unstable angina, transient ischemic attacks and strokes, peripheralvascular disease, arterial thrombosis, preeclampsia, embolism,restenosis following angioplasty, carotid endarterectomy, andanastomosis of vascular grafts; wherein the method comprisesadministering to said human a therapeutically-effective amount of anovel spiro compound of this invention.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “spiro” refers to a compound consisting of two rings havingonly one carbon atom in common. Spiropentane is an exemplary compoundhaving a spiro system. Spiro systems exclude other bicyclic compoundssuch as naphthalene which have two or more carbon atoms in common.

The term “alkyl” used herein refers to a monovalent straight or branchedchain radical of from one to ten carbon atoms, including, but notlimited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-hexyl, and the like.

The term “halosubstituted alkyl” as used herein refers to an alkyl groupas just defined, substituted by one, two or three halogen atoms selectedfrom fluorine, chlorine, bromine, and iodine. Examples of such groupsinclude chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term “aryl” when used alone means a homocyclic aromatic radicalwhether or not fused. Preferred aryl groups include phenyl, naphthyl,biphenyl, phenanthrenyl, naphthacenyl, and the like.

The term “substituted aryl” denotes an aryl group substituted with one,two, or three substituents chosen from halogen, hydroxy, protectedhydroxy, cyano, nitro, C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, trifluoromethyl,amino, aminomethyl, and the like. Examples of such groups are4-chlorophenyl, 2-methylphenyl, 3-methyl-4-hydroxyphenyl, and3-ethoxyphenyl.

The term “arylalkyl” means one, two or three aryl groups having thenumber of carbon atoms designated, appended to an alkyl radical havingthe number of carbon atoms designated. A typical arylalkyl group is thebenzyl group.

The term “alkenyl” as used herein refers to a monovalent straight orbranched chain radical of from two to six carbon atoms containing acarbon double bond including, but not limited to, 1-propenyl,2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl and the like.

The term “alkylene” as used herein refers to a divalent straight orbranched chain group of from one to ten carbon atoms, including but notlimited to, —CH₂—, —(CH₂)₂—. —(CH₂)₃—, —CH(CH₃)—, —CH(C₂H₅)—,—CH(CH₃)CH₂—, and the like.

The term “alkenylene” as used herein refers to a divalent straight orbranched chain group of from two to ten carbon atoms containing acarbon-carbon double bond, including but not limited to, —CH═CH—,—C(CH₃)═CH—, CH═CH—CH₂—, —CH═C(CH₃)—CH₂—, —CH₂CH(CH═CH₂)CH₂, and thelike.

The term “alkynylene” as used herein refers to a divalent straight orbranched chain group of from two to ten carbon atoms containing acarbon-carbon triple bond, including but not limited to,

and the like.

The term “amidino” refers to the radical having the structural formula;

The term “basic radical” refers to an organic radical which is a protonacceptor. Illustrative basic radicals are amino and amidino. Basicradicals may also be formed from a ring nitrogen.

The term “basic group” refers to an organic group containing one or morebasic radicals. A basic group may comprise only an basic radical.

The term “acid radical” refers to an organic radical which is a protondonor. Illustrative acid radicals include;

The term “acidic group” is an organic group containing one or more acidradicals. An acidic group may comprise only an acid radical.

The term “non-interfering substituent” refers to an organic radicalwhich does not significantly reduce the therapeutic effectiveness of acompound.

COMPOUNDS OF THE INVENTION

This invention provides compounds of the general formula (I), or apharmaceutically-acceptable salt, solvate or or prodrug thereof:

wherein;

the atoms A_(i) and B_(j) are independently selected from carbon,nitrogen, oxygen or sulfur, provided that at least one atom of A_(i) iscarbon, and at least one atom B_(j) is carbon;

the rings of the spirobicycle formed by A_(i) and B_(j), respectively,may optionally be partly unsaturated;

p and q are independently numbers from 2 to 6;

m is a number from zero to p;

R₁₀ is the same or different and is a non-interfering substituentindependently selected from hydrogen, alkyl, halosubstituted alkyl,alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, hydroxy, alkoxy,arylalkoxy, amino, substituted amino, carbamoyl, carboxy, acyl, cyano,halo, nitro, sulfo, ═O, or ═S, with the proviso that only one R₁₀ may be═O or ═S, if p is 2 or one or two R₁₀ may be ═O or ═S, if p is a numberfrom 3 to 6;

n is the number from zero to q;

R₀ is the same or different and is a non-interfering substituentindependently selected from hydrogen, alkyl, halosubstituted alkyl,alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, hydroxy, alkoxy,arylalkoxy, amino, substituted amino, carbamoyl, carboxy, acyl, cyano,halo, nitro, sulfo, ═O, or ═S, with the proviso that only one R₀ may be═O or ═S, if q is 2 or one or two R₀ may be ═O or ═S, if q is a numberfrom 3 to 6;

the linking group —(L)— is a bond or a divalent substituted orunsubstituted chain of from 1 to 10 atoms selected from the groupconsisting of carbon, nitrogen, sulfur, and oxygen;

Q is a basic group containing one or more basic radicals; and

R₃ is an acidic group containing one or more acid radicals.

Compounds of the Invention with Preferred Spriro Nuclei

A compound of formula (II), or a pharmaceutically-acceptable salt,solvate or prodrug thereof:

Q—(L)_(z)—Z—R₃  (II)

wherein Z is a spirocyclic nucleus selected from (A), (B), (C), or (D)below

Nucleus (A)

Nucleus (B)

Nucleus (C)

Nucleus (D)

wherein:

the group Q—(L)_(z)— is bound to the nitrogen containing ring of nuclei(A), (B), (C), or (D) and the group R₃ is bound to the ring formed bythe groups A₄₁, A₄₂, A₄₃, A₅₁, A₅₂, A₅₃, A₅₄, A₆₁, A₆₂, A₆₃, A₆₄, A₆₅,A₇₁, A₇₂, A₇₃, A₇₄, A₇₅, or A₇₆; or

the group R₃ is bound to the nitrogen containing ring and the groupQ—(L)_(z)— is bound to the ring formed by the groups A₄₁, A₄₂, A₄₃, A₅₁,A₅₂, A₅₃, A₅₄, A₆₁, A₆₂, A₆₃, A₆₄, A₆₅, A₇₁, A₇₂, A₇₃, A₇₄, A₇₅, or A₇₆;

r and s are independently a number from zero to 5 with the proviso thatnot both r or s are 0 and (r+s) is not more than 6, and z is zero orone;

atoms A₄₁, A₄₂, A₄₃, A₅₁, A₅₂, A₅₃, A₅₄, A₆₁, A₆₂, A₆₃, A₆₄, A₆₅, A₇₁,A₇₂, A₇₃, A₇₄, A₇₅, or A₇₆ are independently selected from carbon,nitrogen, oxygen or sulfur, provided that at least one of said atoms iscarbon;

the hydrogens of the nitrogen containing part of the spirocycle Z may besubstituted by a number of m substituents R₁₀, wherein;

m is a number from zero to (r+s); and

R₁₀ is the same or different and is a non-interfering substituentindependently selected from alkyl, halosubstituted alkyl, alkenyl,alkynyl, cycloalkyl, aryl, arylalkyl, hydroxy, alkoxy, arylalkoxy,amino, substituted amino, carbamoyl, carboxy, acyl, cyano, halo, nitro,sulfo, ═O, or ═S, with the proviso that only one or two R₁₀ may be ═O or═S;

n is a number from zero to 3 in Z of having nuclei (A), or a number fromzero to 4 in Z having nuclei (B), a number from zero to 5 in Z havingnuclei (C), or a number from zero to 6 in Z having nuclei (D);

R₀ is the same or different and is a non-interfering substituentindependently selected from alkyl, halosubstituted alkyl, alkenyl,alkynyl, cycloalkyl, aryl, arylalkyl, hydroxy, alkoxy, arylalkoxy,amino, substituted amino, carbamoyl, carboxy, acyl, cyano, halo, nitro,sulfo, ═O, or ═S, with the proviso that only one or two R₀ may be ═O or═S; and

Q, L, and R₃ are as defined previously for the formula I compounds.

Representative Spirocyclic Nuclei

The spirocyclic compounds of the invention include spirocyclic nucleiselected from the group represented by the following structuralformulae:

Representative oxo substituted Spirocyclic nuclei are as follows

Oxo substituted spirocyclic compounds of the invention includespirocyclic nuclei selected from the group represented by the followingstructural formulae:

Preferred spirocyclic nuclei include the following nuclei:

where m is one or two.

A group of more preferred spirocyclic nuclei includes the followinggroups:

where m is one or two.

A second group of more preferred nuclei include the following:

where m is one or two.

The most preferred spirocyclic nuclei include teh following nuclei:

where m is one or two.

The Basic Substituent

The substituent Q of formulae (I) and (II) is a basic group. A basicgroup contains one or more basic radicals. Suitable basic radicalscontain one or more nitrogen atoms and include amino, imino, amidino,hydroxyamidino, N-alkylamidines, N,N′-dialkyamidines, N-arylamidines,aminomethyleneamino, iminomethylamino, guanidino, aminoguanidino,alkylamino, dialkylamino, trialkylamino, alkylideneamino, pyrrolyl,imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, indolizinyl,isoindolyl, 3H-indolyl, indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl,isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, amide, thioamide, benzamidino, pteridinyl,4aH-carbozolyl, carbozolyl, beta-carbolinyl, phenanthridinyl, acridinyl,pyrimidinyl, phenanthrolinyl, phenazinyl, phenarsazinyl, phenothiazinyl,pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperidyl, piperazinyl, indolinyl, isoindolinyl, quinuclidinyl,morpholinyl, or any of the preceding substituted with amino, imino,amidino, hydroxyamidino, aminomethyleneamino, iminomethylamino,guanidino, alkylamino, dialkylamino, trialkylamino,tetrahydroisoquinoline, dihydrosioindole, alkylideneamino groups or agroup represented by the formula;

Preferred basic radicals are selected from amino, piperidyl, guanidino,hydroxyamidino, and amidino. The most preferred basic radicals areamidino, hydroxyamidino, and piperidyl represented by the formulae;

The basic group and linker, Q—(L)_(z)—, may have the form of a basicradical pendant on a cyclic ring as shown in formula IV below. The D ofFormula (IV) ring may also have substituents R₂₀ which are selected fromchlorine, fluorine or non-interfering organic radicals. Fluorine ispreferred as a substituent on the D ring. The R₂₀ substituents may be tin number, where t is an integer from zero to the number of unsatisfiedbonds in the D ring. The basic radical(s) attaches to the D ring in themanner shown in formula (IV) below:

Suitable D rings are formed from a nucleus selected from the groupconsisting of; benzene, cycloheptadiene, cycloheptatriene, cycloheptane,cyclohexane, cyclohexene, cyclohexadiene, cycloheptene, cyclooctadiene,cyclooctane, cyclooctatetraene, cyclooctene, cyclopentane, cyclopentene,imidazole, isooxazole, morpholine, oxazole, piperazine, piperidine,pyrazine, pyrazole, pyridine, pyrimidine, pyrrole, pyrrolidine,pyrroline, tetrahydropyridine, tetrahydropyrimidine, 1H-tetrazole,thiazolidine, thiazole, thiopyran, 1,3,5-triazine, 1,2,3-triazole,1,2,4-triazole, dihydrofuran, dihydropyran, dioxane, dioxepin,dioxolane, furan, oxocane, tetrahydrofuran, tetrahydropyran, thiophene,and tetrahydrothiophene.

An illustrative species of formula (IV) is the basic radical attached toa benzene ring as shown in formulae (V) and (VI) below:

The preferred basics groups include the following groups:

wherein R₂₀ is hydrogen or halogen.

The Acidic Substituent R₃

The substituent R₃ of formula (I) or (II) is an acidic group. An acidicgroup contains one or more acidic radicals. Suitable acidic radicalscontain one or more proton donors, and include groups such as sulfonicacids, tetrazoles, phosphonic acids, carboxylic acids, and the like. Theacidic radical may be bound to an aryl group, such as phenyl orsubstituted phenyl, or bound to alkyl chains, such as methylene. Thesegroups may also be bound to the spirocyclic nucleus through alkyl chainshaving heteroatoms, suchs as S, O, or N, and amide (CONH) or carbonyl(CO) groups. The acidic substituent may also comprise an α-sulfonamidocarboxylic acid group of the formula:

Preferred Spiro Compounds are as Follows

The formula II compounds, a preferred subset of the formula Ispirocyclic compounds, which are preferred compounds of the inventioninclude the following compounds, their pharmaceutically-acceptablesalts, solvates, and prodrug derivatives, as follows:

wherein X is F or H, m is zero to four, and n is one to four.

A more preferred subset of the formula I spirocyclic compounds includethe following compounds, their pharmaceutically-acceptable salts,solvates, and prodrug derivatives, as follows:

wherein X is F or H, m is zero to four, and n is one to four.

A second preferred subset of the formula I spirocyclic compounds includethe following compounds, their pharmaceutically-acceptable salts,solvates, and prodrug derivatives, as follows:

wherein X is F or H.

A second more preferred subset of the formula I spirocyclic compoundsinclude the following compounds, their pharmaceutically-acceptablesalts, solvates, and prodrug derivatives, as follows:

wherein X is F or H.

A most preferred subset of the formula I spirocyclic compounds includethe following compounds, their pharmaceutically-acceptable salts,solvates, and prodrug derivatives, as follows:

wherein X is F or H, m is zero to four, and n is one to four.

A second most preferred subset of the formula I spirocyclic compoundsinclude the following compounds, their pharmaceutically-acceptablesalts, solvates, and prodrug derivatives, as follows:

wherein X is F or H.

The compounds of the invention possess at least one acidic functionalsubstituent (viz., R₃ of Formula I or II) and, as such, are capable offorming salts. Representative pharmaceutically-acceptable salts, includebut are not limited to, the alkali and alkaline earth salts such aslithium, sodium, potassium, calcium, magnesium, aluminum and the like.Salts are conveniently prepared from the free acid by treating the acidin solution with a base or by exposing the acid to an anion exchangeresin on the salt cycle.

Included within the definition of pharmaceutically-acceptable salts arethe relatively non-toxic, inorganic and organic base addition salts ofcompounds of the present invention, for example, ammonium, quaternaryammonium, and amine actions, derived from nitrogenous bases ofsufficient basicity to form salts with the compounds of this invention(see, for example, S. M. Berge, et. al., “pharmaceutical Salts,” J.Phar. Sci., 66: 1-19 (1977)).

The basic portion of the compounds of the invention (viz., part Q offormula I or II) may be reacted with suitable organic or inorganic acidsto form salts of the invention. Representative salts include thoseselected from the group comprising; acetate, benzenesulfonate, benzoate,bicarbonate, bisulfate, bitartrate, borate, camsylate, carbonate,chloride, clavulanate, dihydrochloride, edetate, edisylate, estolate,esylate, fumarate, gluceptate, gluconate, glutamate,glycollylarsanllate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, malseate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,oleate, oxalate, palmitate, pantothenate, phosphate, polygalacturonate,salicylate, stearate, subacetate, succinate, tannate, tartrate,tosylate, trifluoroacetate, trifluoromethane sulfonate, and valerate.

The compounds of the formula (I) or (II) can also be in the form ofzwitterions, since they contain both acidic and basic functionality andare capable of self-protonation.

Certain compounds of the invention possess one or more chiral centersand may thus exist in optically active forms, or as mixtures ofdiastereomers. Likewise, when the compounds contain an alkenyl oralkenylene group there exists the possibility of cis- and trans-isomeric forms of the compounds. The R- and S- isomers and mixturesthereof, including racemic mixtures as well as mixtures of cis- andtrans- isomers, are contemplated by this invention. Additionalasymmetric carbon atoms can be present in a substituent group such as analkyl group. All such isomers as well as the mixtures thereof areintended to be included in the invention. If a particular stereoisomeris desired, it can be prepared by methods well known in the art by usingstereospecific reactions with starting materials which contain theasymmetric centers and are already resolved or, alternatively by methodswhich lead to mixtures of the stereoisomers and subsequent resolution byknown methods.

Prodrug Derivatives of Compounds of the Invention

Prodrugs are derivatives of the compounds of the invention which havemetabolically cleavable groups and become by solvolysis or underphysiological conditions the compounds of the invention which arepharmaceutically active in vivo. For example, ester derivatives ofcompounds of this invention are often active in vivo, but not in vitro.Other derivatives of the compounds of this invention have activity inboth their acid and acid derivative forms, but the acid derivative formoften offers advantages of solubility, tissue compatibility, or delayedrelease in the mammalian organism (see, Bundgard, H., Design ofProdrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs includeacid derivatives well known to practitioners of the art, such as, forexample, esters prepared by reaction of the parent acid with a suitablealcohol, or amides prepared by reaction of the parent acid compound withan amine. Simple aliphatic or aromatic esters derived from acidic groupspendant on the compounds of this invention are preferred prodrugs. Insome cases it is desirable to prepare double ester type prodrugs such as(acyloxy)alkyl esters or ((alkoxycarbonyl)oxy)alkyl esters.

Preferred are the C₁-C₈ alkyl, C₂-C₈ alkenyl, aryl, C₇-C₁₂ substitutedaryl, and C₇-C₁₂ arylalkyl esters of the compounds of the invention (performula I or II) Particularly preferred are the C₁-C₄ alkyl esters, forexample, where the R₃ acidic group has been esterified to form a grouprepresented by one of the following formulae:

Acylated basic radicals which are part of basic group on the compoundsof the invention have been found to significantly enhancebioavailability. Without being bound by any theory of operation, it isbelieved that lowering the basicity of basic group (Q) makes thecompounds of this invention less subject to “food effect”, that is, theyhave good availability in therapeutic administration to an animalwithout fasting.

Compounds of this invention may beneficially be dual prodrugderivatives. For example, the acidic group (R₃) may be reacted to forman ester and the basic group may additionally be reacted to form anacylated basic derivative. The prodrug derivatives of the compounds ofthis invention may be combined with other features herein taught toenhance bioavailability, for example, substitution of fluorine atoms onthe basic benzamidine group.

Another highly preferred class of prodrugs of the invention are thoseformed by acylating the basic radicals present on the compounds of theinvention. The acyl portion of the acylated basic radical has thegeneral formula:

where R is C₁-C₈ alkyl, C₂-C₈ alkenyl, aryl, C₇-C₁₂ substituted aryl,and C₇-C₁₂ arylalkyl; and X is a bond, O, S, or N. Preferably R is C₁-C₄alkyl and X is oxygen. For example, acylated basic radical prodrugs ofthe invention are prepared and illustrated in A, B, C, and D below:

 A) acylation of amidine results in a prodrug derivative group:

 B) acylation of a cyclic amine such as piperidine results in a prodrugderivative group:

C) acylation of guanidine results in a prodrug derivative group:

D) acylation of a primary amine results in a prodrug derivative group:

where, for A, B, C, and D above, R and X are as defined above for theacylated portion of the basic group.

Preparation of Spiro Compounds

The synthesis of spiro compounds covered by the invention is describedin Scheme 1 thru Scheme 11, in which the following terms are used:

P means a general protective group for amines like benzyl,tert.-butoxycarbonyl, benzyloxycarbonyl, or ethoxycarbonyl.

X, when present, is a spacer typically consisting of a chain of up tothree carbon atoms, e.g. methylene, dimethylene, or trimethylene.

Scheme Nomenclature

The substituent R is a non-interfering substituent illustrated by analkyl group selected from ethyl, methyl, or tert.-butyl forming esterscontaining the group COOR, which are cleaved to the correspondingcarboxylic acids (R═H).

Scheme 1 describes the synthesis of1-oxa-3,8-diaza-spiro[4.5]decan-2-ones (see J. M. C aroon, R. D; Clark,A. F. Kluge, J. T. Nelson, A. M. Strosberg, St. H. Unger, A. D. Michel,R. L. Whiting, J. Med. Chem. 1981, 24, 1320). 4-Piperidinone isN-protected, e.g. by reaction with benzyl chloroformate, and thiscompund is converted to the shown epoxide by addition of a methylenegroup using trimethylsulfoxonium iodide/sodium hydride in DMSO. The ringopening of the epoxide requires heating with an excess of4-cyanoaniline, and the following formation of the spiro-oxazolidinoneis achieved with N,N′-carbonyl diimidazole, diethyl carbonate, or withphosgene. After removal of the protective group the piperidine isalkylated with an ω-halogenocarboxylate like ethyl bromoacetate or ethyl4-bromobutanoate. Finally, the nitrile is converted to the amidine byreaction with ethanolic hydrochloric acid followed by treatment withammonia, and the ethyl ester is cleaved under mild aqueous basicconditions to give the amidino carboxylic acid.

The synthesis of 3-phenyl-1-oxa-2,8-diaza-spiro[4.5]dec-2-enes isoutlined in Scheme 2. 4-Methylene-piperidines like1-(tert.butoxycarbonyl)-4methylene-piperidine are prepared from thecorresponding N-protected piperidinones by Wittig reaction. Thefive-membered ring is formed by addition of 4-cyanobenzonitrile oxide,which is generated in situ from 4-cyanobenzohydroximinoyl chloride withtriethylamine (see K.-C. Liu, B. R. Shelton, R. K. Howe, J. Org. Chem.1980, 45, 3916; L. Fisera, F. Sauter, J. FrÖhlich, Y. Feng, P. Ertl, K.Mereiter, Monatshefte Chem. 1994, 125, 553). The protective group isremoved with trifluoroacetic acid followed by alkylation of thespiropiperidine and conversion of the nitrile to the amidine asdescribed in the previous Scheme.

The synthesis of 2,4-dioxo-1,3,8-triaza-spiro[4.5]decane derivatives isdescribed in Scheme 3. N-protected piperidin-4-ones like1-benzyloxycarbonyl-piperidin-4-one are converted to the correspondingspirohydantoins by heating with a mixture of potassium cyanide andammonium carbonate (see G. Winters, V. Aresi, G. Nathansohn, Farmaco,Ed. Sci. 1970, 25, 681). The protective group is removed byhydrogenation, and the piperidine is treated with 4-cyanobenzoylchloride. Alkylation of this intermediate with ω-halogenoalkanoatesgives the shown 3-substituted derivatives, and in a second alkylationstep the nitrogen at position 1 may be alkylated with alkyl halidesR'Hal, e.g. with benzyl bromide leading to1-benzyl-2,4-dioxo-1,3,8-triaza-spiro[4.5]decanes (see O. O. Orazi, R.A. Corral, H. Schuttenberg, J. Chem. Soc., Perkin Trans. I, 1974, 219).The preferred method for conversion of the benzonitrile to an amidineemploys the sequence of addition of hydrogen sulfide, alkylation of theintermediate primary thioamide with methyl iodide, heating with ammoniumacetate, and purification of the crude amidine after protection withtert.-butoxycarbonyl. In a final step the protective group is removedwith trifluoroacetic acid. If the acidic side chain has been masked byan tert.-butyl ester this one is also cleaved under these conditions.

In an alternative sequence 1-substituted2,4-dioxo-1,3,8-triaza-spiro[4.5]decane derivatives may be prepared asshown in Scheme 4 (see G. M. Carrera, Jr., D. S. Garvey, J. HeterocyclicChem. 1992, 29, 847). The piperidone is treated with a mixture ofpotassium cyanide and a primary amine like benzyl amine. Theintermediate 4-amino-4-cyanopiperidine is hydrolyzed followed by ringclosure with potassium cyanate. The following steps including thealkylation of the nitrogen in position 3 with ω-halogenoalkanoates andformation of the amidine is carried out as described in Scheme 4.

Scheme 5A describes the synthesis of1,3-dioxo-2,8-diaza-spiro[4.5]decane derivatives (see E. Jucker, R.SüβArch. Pharm. 1961, 294/66, 210; Helv. Chim. Acta 1977, 49, 1135; Y.Ishihara, H. Yukimasa, M. Miyamoto, G. Goto, Chem. Pharm. Bull. 1992,40, 1177). The shown 2-cyanoacrylate is formed by Knoevenagelcondensation between N-protected piperidin-4-ones likeN-benzylpiperidinone and ethyl cyanoacetate. It is heated with potassiumcyanide in ethanol/water followed by hydrolysis with hydrochloric acid.The diacid may be purified by reesterification followed by anotherhydrolysis step of the substituted diethyl succinate, and the succinicacid is converted to the spiro succinic anhydride with dehydrationreagents like dicyclohexylcarbondiimide (DCC) or acetanhydride. It istreated in situ with ω-aminoalkanoates to give the spiroimides. Afterremoval of the protective group the reaction with 4-cyanobenzoylchloride, formation of the amidine and saponification of the ester arecarried out as described before.

In a similar sequence 2-phenyl-1,3-dioxo-2,8-diaza-spiro[4.5]decanes areprepared. In contrast to Scheme 5A the intermediate succinic anhydridereacts with 4-aminobenzonitrile, and after deprotection of thespiropiperidine the intermediate is alkylated with ω-halogenoalkanoates.The final steps of the synthesis are carried out in the usual manner.

Scheme 6 illustrates the synthesis of2-phenyl-1,3-dioxa-8-aza-spiro[4.5]decanes. N-protected4-methylenepiperidine epoxides like benzyl1-oxa-6-aza-spiro[2.5]octane-6-carboxylate are hydrolyzed to4-hydroxy-4-hydroxymethylpiperidines by heating with diluted aqueoushydrochloric acid (see Eur. Pat. Appl. EP 189 370). The spiro-bicycle isobtained by condensation of the diol with 4-cyanobenzaldehyd, which maybe achieved by heating in toluene and catalytic amounts of4-toluenesulfonic acid or by reaction in the presence of borontrifluoride. The following steps: removal of the protecting group,alkylation with ω-halogenoalkanoates, formation of the amidine, andhydrolysis of the ester; are carried out as described in the previousSchemes.

Scheme 7 describes the synthesis of8-phenyl-1-oxa-3,8-diaza-spiro[4.5]decan-2-ones.1-(4-Cyanophenyl)piperidin-4-ol is prepared by heating a mixture of4-chlorobenzonitrile and piperidin-4-ol in DMF in the presence of sodiumcarbonate. The following oxidation to the piperidone is achieved withDMSO/oxalyl chloride (A. J. Mancuso, D. Swern, Synthesis 1981, 165), andthe epoxide is formed by reaction with trimethylsulfoxoniumiodide/sodium hydride in DMSO. After ring opening by reaction withω-aminoalkanoates the following steps of formation of the Spiroderivative and the amidino acid are carried out according to methodsdescribed in Scheme 1.

The synthesis of 1-oxa-4,9-diaza-spiro[5.5]undecan-3-ones is describedin Scheme 8A (see R. D. Clark, J. M. Caroon, D. B. Repke, A. M.Strosberg, S. M. Bitter, M. D. Okada, A. D. Michel, R. L. Whiting, J.Med. Chem. 1983, 26, 855). N-protected 4-methylenepiperidine epoxideslike benzyl 1-oxa-6-aza-spiro[2.5]octane-6-carboxylate are opened byheating with a methanolic solution of ammonia to give the corresponding4-aminomethyl-4-hydroxypiperidine. The spiro-bicyclic nucleus is formedby the following condensation with chloroacetyl chloride. After removalof the benzyloxycarbonyl group with HBr in acetic acid thespiropiperidine is acylated with 4-cyanobenzoyl chloride. The subsequentsteps of alkylation with ω-halogenoalkanoates, formation of the amidine,and cleavage of the ester are carried out as described in the previousSchemes.

Scheme 8B illustrates the synthesis of the related1-oxa-4,9-diaza-spiro[5.5]undecanes. These are prepared by reduction ofN(9)-protected 1-oxa-4,9-diaza-spiro[5.5]undecan-3-ones with lithiumaluminum hydride. After alkylation with ω-halogenoalkanoates theprotective group is removed, and the final steps of the sequence arecarried out according to Scheme 8A.

The synthesis of 1,3,8-triaza-spiro[4.5]dec-1-en-4-ones is described inScheme 9 (see C. A. Bernhart, et al., J. Med. Chem. 1993, 36, 3371; C.del Campo, E. F. Llama, Org. Prep. Proced. Int. 1990, 22, 514).Protected 4-aminopiperidine-4-carboxamides are prepared by addition ofpotassium cyanide to corresponding piperidin-4-ones likeN-benzylpiperidin-4-one followed by hydration of the nitrileintermediate. The spiro-bicyclic nucleus is obtained by heating withtriethyl orthoformate or by reaction with gaseous formaldehyde. It isalkylated at the nitrogen in position 3 with ω-halogenoalkanoates andthe benzyl group is removed by hydrogenation. After acylation with4-cyanobenzoyl chloride the amidinocarboxylic acid is prepared accordingto methods described in the previous Schemes.

Scheme 10 describes the synthesis of (3-aza-spiro[5.5]undec-9-yl)aceticacid derivatives. After protection of ethyl isonipecotate with benzylchloroformate the ester is reduced with lithiumaluminum hydride followedby Swern oxidation with oxalyl chloride/DMSO to corresponding4-formylpiperidine. The Spiro derivative is formed by condensation with1-buten-3-one under basic conditions using potassium hydroxide, and theside chain is introduced by Horner—Emmons reaction with diethyl(ethoxycarbonyl)methylphosphonate/sodium hydride. The bicyclic nucleusand the exocyclic double bond are saturated and the protective group isremoved by catalytic hydrogenation with palladium hydroxide on charcoal.The subsequent acylation with 4-cyanobenzoyl chloride, formation of theamidine, and hydrolysis of the ethyl ester are carried out by methodsdescribed in the previous Schemes.

Scheme 11a describes the preparation of 6,5 spiro-fusedpiperidino-carbamates in which the carbamate nitrogen is substitutedwith an acetic acid residue and the piperidine nitrogen is acylated witha benzamidine. In the first step, 4-piperidone (1) is allowed to reactwith TMSCN resulting in the formation of cyanohydrin (3). The nitrilemoiety is reduced with LAH providing amino-alcohol (5), which is thenallowed to react with diethyl carbonate in the presence of NaHultimately forming spiro carbamate (7). Alkylation of the carbamatenitrogen is accomplished with NaH and alpha-bromo acetate giving ester(9). Catalytic hydrogenation removes the benzyl group providing freeamine (11) which is acylated with 4-cyanobenzoic acid yielding (13). Thenitrile moiety in (13) is converted to a protected amidine and isisolated as its Boc derivative. This material is then fully deprotectedwith TFA providing (15) as a salt.

Materials containing a 6,6 spiro-fused piperidino-carbamate can beprepared in an analogous fashion (see Scheme 11b). Lithio-acetonitrileis allowed to react with 4-piperidone resulting in the formation ofalcohol (17). This material can be transformed into final product (23)using the same set of reactions described for the conversion of (1) to(15).

Scheme 11c describes the preparation of compounds containing adisubstituted 6,6 and 6,5 spirolactone. In the first step, piperidone 25is allowed to react with the olefinic grignard reagent giving adduct 27.Oxidation of this material with permanganate affords lactone 29.Alkylation of the lactone enolate with an αbromo ester provides thefunctionalized product 31. Removal of the Boc protecting group with TFAliberates amine 33 which can be acylated with 4-cyanobenzoic acidproviding adduct 35. The nitrile moiety in this molecule can beconverted to an amidine using the thio-Pinner protocol and thendeprotected giving compound 39.

Scheme 12 describes the preparation of 3,9-diazaspiro-[5.5]undecanecompounds. The diazaspiro skeleton is prepared according to knownprocedures (see S. M. McElvain and R. E. Lyle, Jr., J. Am. Chem. Soc.,1967, 32, 1966). The intermediate diazasprirocyclic compound isalkylated by ω-haloalkanoodes. After removal of the benzyl group, it isalkylated either by N-protected-4-(ω-haloalkyl)piperidines or bycorresponding pyridines, which are readily reduced to piperidines byhydrogenation as shown in the experimental section below. In a similermanner, any other basic residue with an appropriate spaced is prepared.

1-Monoesters of aspartic acid (n=1) or glutamic acid (n=2), N-protectedderivatives of these monoesters, or other similarly protected compoundsare commercially available or have been described previously in theliterature. See Gregory et al., J. Chem. Soc. (c) , 1968, 715; Olsen etal., J. Org. Chem., 1984, 49, 3527; Yang and Menifield, J. Org. Chem.,1976, 41, 1032; Taschner et al., Liebigs Ann. Chem., 1961, 646, 123,125, 127, 134. These compounds are connected to sulfonamides by reactionwith sulfonyl chlorides. Preferred sulfonyl chlorides arebenzenesulfonyl chloride or n-butanesulfonyl chloride. The3,9-diazaspiro-[5.5]undecane compound from Scheme 12 is acylated bythese intermediates, followed by removal of the portective group. It isacylated by N-protected ω-aminocarboxylic acids bearing protectivegroups, such as t-butoxycarbonyl or benzyloxycarbonyl. The protectivegroup is removed and the free amine is converted to a guanidine, whichis optionally protected using standard procedures such as by reactionwith N,N′-bis(t-butoxycarbonyl)thiourea and heavy metal salts (e.g.copper or mercury).

Scheme 14 outlines the preparation of the 6,5 (n=1) and 6,6 (n=2)spiro-carbamates with substitution of a basic residue on the piperidinenitrogen and an acidic residue on the carbamate nitrogen. The protectedcompound is deprotonated with sodium hydride in an aprotic solvent, suchtetrahydrofuran, and the resulting sodium salt is reacted with an α-haloester to provide the mono-substituted products. These compounds are thendeprotected to give the secondary amines. These materials are thenacylated giving N-alkylated, N-acylated intermediate products. Thenitrile group is transformed to an amidino group using a modifiedthio-Pinner sequence. More specifically, reaction of the nitrile to formthe thioamide followed by S-alkylation with methyl iodide, and thendisplacement with ammonia. Preferably, the intermediate compounds arenot isolated but are reacted with di-t-butyl dicarbonate to give theprotected amidines. These intermediate compounds are fully deprotected,for example with trifluoroacetic acid, to give the desired products.

Scheme 15 outlines the preparation of 6,5 spiro-carbamates withsubstitution of an alkyl guanidine on the piperidine nitrogen, and anacicic residue on the carbamate nitrogen. The intermediate compoundprepared as described in Scheme 14, is acylated with protected aminoacids providing the intermediate amide compounds. The protecting groupsare then removed using reactions well known in the chemical arts, andmaterials are guanylated providing the fully-protected intermediatecompounds. Complete deprotection, for example with trifluorocaceticacid, provides the desired compounds.

Scheme 16 describes the synthesis of spiro-carbamates containing anoxygen-linked basic group, where n is 1 or 2. The starting compound isalkylated on nitrogen by reacting with a strong base, such as sodiumhydride, and an alkylating agent, such as an α-bromo-t-butyl acetate.The protecting group for the ketone functional group is then removed,and the ketone reduced with a hydride reducing agent, such as sodiumborohydride, to provide a mixture of alcohols. The alcohols arealkylated with 4-cyanobenzyl bromide, and the resulting compound istransformed into the protected amidine.

Scheme 17 describes the synthesis of spiro carbamates containing anamide-linked basic group on the saturated ring. The intermediate ketone,prepared as shown in Scheme 16, is reductively aminated with an amineand sodium cyanoborohydride. This intermediate compound is acylated with4-cyanobenzoic acid providing the amide intermediate. This amide isconverted to the protected amidine using procedures well known in thechemical arts. The resulting compound is converted into the desiredcompound using procedures as described previously.

Scheme 19 describes the synthesis of9-benzoyl-2,9-diaza-spiro[5.5]undecanes in which the nitrogen in the2-position is acylated. The 2,9-diaza-spiro[5.5]undecane template isprepared as described in Example 40 (see also U.S. Pat. No. 5,451,578).Treatment of this mono-protected material with 4-cyanobenzoyl chloride(or 2-fluoro-4-cyanobenzoyl chloride) in the presence of a base such astriethylamine gives the benzamide intermediate. The protecting group isremoved with trifluoroacetic acid followed by acylation with theappropriate ester-acid chloride such as methyl oxalyl chloride or methyladipoyl chloride. After mild basic hydrolysis of the ester, the nitrileis converted to the amidine utilizing a three step protocol: 1)treatment with hydrogen sulfide in pyridine in the presence oftriethylamine; 2) treatment with methyl iodide in acetone; and 3)treatment with ammonium acetate in ethanol, thus providing the amidinocarboxylic acids.

Scheme 20 describes the synthesis of9-benzoyl-2,9-diaza-spiro[5.5]undecanes in which the nitrogen in the2-position is alkylated. After removal of the protecting group withtrifluoroacetic acid in the benzamide intermediate as in Scheme 19, thesecondary amine is alkylated with the appropriate halogenoester such asethyl bromoacetate or ethyl 4-bromobutanoate. Mild basic hydrolysis ofthe ester followed by nitrile to amidine conversion as described in theprevious Scheme provides the amidino carboxylic acid.

Scheme 21 describes the synthesis of2-benzoyl-2,9-diaza-spiro[5.5]undecanes in which the nitrogen in the9-position is acylated. The mono-protected 2,9-diaza-spiro[5.5]undecaneis acylated with the appropriate ester-acid chloride in the presence oftriethylamine. Removal of the protecting group with trifluoroacetic acidis followed by acylation with 4-cyanobenzoyl chloride (or2-fluoro-4-cyanobenzoyl chloride). Mild basic hydrolysis of the ester isfollowed by conversion of the nitrile to the amidine as previouslydescribed for Scheme 19.

Scheme 22 describes the synthesis of2-benzoyl-2,9-diaza-spiro[5.5]undecanes in which the nitrogen in the9-position is alkylated. The mono-protected 2,9-diaza-spiro[5.5]undecaneis alkylated with the appropriate halogenoester in the presence oftriethylamine. Removal of the protecting group with trifluoroacetic acidis followed by acylation with 4-cyanobenzoyl chloride (or2-fluoro-4-cyanobenzoyl chloride). Mild basic hydrolysis of the ester isfollowed by conversion of the nitrile to the amidine as previouslydescribed for Scheme 19.

Scheme 23 describes the synthesis of the8-benzoyl-2,8-diaza-spiro[5.4]decanes in which the nitrogen in the2-position is acylated. The mono-protected 2,9-diaza-spiro[5.5]undecanenucleus (see: J. Med. Chem. 1995, 38, 3772-3779) is acylated with4-cyanobenzoyl chloride (or 2-fluoro-4-cyanobenzoyl chloride).Hydrogenolysis of the benzyl protecting group with palladium on charcoalis followed by acylation with the appropriate ester-acid chloride.Subsequent hydrolysis under mildly basic conditions and then conversionof the nitrile to the amidine as previously described affords theamidino carboxylic acids.

Scheme 24 describes the synthesis of the8-benzoyl-2,8-diaza-spiro[5.4]decanes in which the nitrogen in the2-position is alkylated. Hydrogenolysis of the benzyl protecting groupwith palladium on charcoal is followed by alkylation with theappropriate halogenoester. The amidino carboxylic acids are obtainedupon hydrolysis of the ester and then conversion of the nitrile, aspreviously described, to the amidine.

Scheme 25 describes the synthesis of the2-benzoyl-2,8-diaza-spiro[5.4]decanes in which the nitrogen in theB-position is acylated. The mono-protected 2,8-diaza-spiro[5.4]decane isacylated with the appropriate ester-acid chloride. Hydrogenolysis of thebenzyl protecting group with palladium on charcoal is followed byacylation with 4-cyanobenzoyl chloride (or 2-fluoro-4-cyanobenzoylchloride). Subsequent hydrolysis of the ester under mildly basicconditions and then conversion of the nitrile to the amidine aspreviously described affords the amidino carboxylic acids.

Scheme 26 describes the synthesis of the2-benzoyl-2,8-diaza-spiro[5.4]decanes in which the nitrogen in the8-position is alkylated. The mono-protected 2,8-diaza-spiro[5.4]decaneis alkylated with the appropriate halogenoester. Hydrogenolysis of thebenzyl protecting group with palladium on charcoal is followed byacylation with 4-cyanobenzoyl chloride (or 2-fluoro-4-cyanobenzoylchloride). Hydrolysis of the ester under mildly basic conditions andthen conversion of the nitrile to the amidine as described in Scheme 19affords the amidino carboxylic acids.

The Scheme 27 describes the synthesis of(3-aza-spiro[5.5]undec-9-yl)acetic acid derivatives. Theethylisonipecotate was protected with benzyl chloroformate, then theester was reduced with lithium aluminum hydride followed by swernoxidation with oxalyl chloride/dimethyl sulfoxide to the corresponding4-formylpiperidine. The spirocyclic ring was formed by the basecatalyzed michael addition of methyl vinyl ketone to the aldehydefollowed by acid catalyzed aldol cyclization and dehydration to affordthe desired spirocyclic enone. The side chain elongation is carried outby Horner-Emmons condensation with triethylphosphonoacetate/sodiumhydride in THF. The bicyclic nucleus and the exocyclic double bond aresaturated and the protective group is removed by catalytichydrogenation.

The nitrogen of the aza spirocompound was acylated with p-cyanobenzoylchloride. The nitrile was converted to N-hydroxyamidino by treating withhydroxylamine hydrochloride in triethylamine and ethanol as solvent. Thehydroxylamidino moiety was hydrogenated with 5% Pd/C (50% wet) at 60° C.using 50 psi H₂ overnight to afford the desired amidino functionality.The catalyst was filtered through celite and solvent evaporated underreduced pressure. The hydrolysis of ester was carried out under basicconditions to give the desired final product.

The Scheme 28 describes the synthesis of(2-aza-spiro[5.5]undec-9-yl)acetic acid derivatives. The3-piperidinemethanol was protected with benzylchloroformate and thenoxidized under swern conditions. The subsequent spiro ring formation,Horner-Emmons, acylation, formation of amidine, and hydrolysis of esterwere carried out by methods described in previous Scheme 27.

The Scheme 29 describes the synthesis of(3-aza-spiro[5.5]undec-9-yl)propionic acid derivatives. The spirocyclicenone was synthesized as shown in Scheme37, followed by Horner-Emmonsreaction with triethylphosphono propionate. The bicyclic nucleus and theexocyclic double bond are saturated and the protective group is removedby catalytic hydrogenation.

The nitrogen of the aza spirocompound was acylated with p-cyanobenzoylchloride. The nitrile was converted to N-hydroxyamidino by treating withhydroxylamine hydrochloride in triethylamine and ethanol as solvent. Thehydroxylamidino moiety was hydrogenated with 5% Pd/C (50% wet) at 60° C.using 50 psi H₂ overnight to afford the desired amidino functionality.The catalyst was filtered through celite and solvent evaporated underreduced pressure. The hydrolysis of ester was carried out under basicconditions to give the desired final product.

The Scheme 30 describes the synthesis of(3-aza-spiro[5.5]undec-9-yl)formic acid) derivatives (see U.S. Pat. No.5,451,578). The shown diester is formed by Knoevenagel condensationbetween N-benzylpiperidone and ethyl cyanoacetate. The hydrolysis andesterification is carried out with ethanol and sulfuric acid to give thediester. The diester after purification is reduced to diol with LAH. Thedebenzylation followed by Boc-protection was carried out in one step byhydrogenation with palladium hydroxide in presence of Boc2O. The diolwas converted to mesylate, followed by condensation with diethylmalonateto afford the spirocyclic diester. The diester was hydrolyzed to diacidfollowed by decarboxylation to afford the3-aza-spiro[5.5]undec-9-yl)formic acid. The deprotection with TFAfollowed by acylation with p-cyanobenzoyl chloride and conversion ofnitrile to amidino was carried out utilizing a three step protocol: 1)treatment with hydrogen sulfide in pyridine in the presence oftriethylamine; 2) treatment with methyl iodide in acetone; and 3)treatment with ammonium acetate in ethanol, thus providing the amidinocarboxylic acid.

The Scheme 35 describes the synthesis3-{2-[(3-Azaspiro[5.5]undecane-9-carbonylamino} acetic acid. TheBOC-acid from Scheme 34 is coupled with glycine ethyl ester or3-aminoethylpropionate in presence EDC, HOBt, DIEA to afford the amide.The deprotection of Boc followed by acylation with p-cyanobenzoylchloride, amidino formation, and hydrolysis of ester are carried out asdescribed in the previous schemes.

The Scheme 32 describes the synthesis of8-benzoyl-1,3,8-triaza-spiro[4.5]dec-1-en. The spiro-imidazolinetemplate is synthesized from N-Boc-4-piperidone, via synthesis of aminonitrile by the strecker reaction. Reduction of amino nitrile with LAH toethylene diamine. Ethylene diamine is cyclized by mild reaction withformamidine acetate in ethanol at room temperature. The alkylation ofspiro-imidazoline with appropriate halogenated alkyester gives mixtureof N-1 and N-3 alkylated product which can be separated. The protectinggroup is removed with trifluroacetic acid and N-8 is acylated with4-cyanobenzoyl chloride. After basic hydrolysis of ester, the nitrile isconverted to amidine utilizing the procedure as described in earlierschemes.

The Scheme 33 describes the synthesis of 8-aza-spiro[5.4]dec-3-en-2-onein which the nitrogen in the 8-position is alkylated or acylateddepending of the derivative being synthesized. The intermediateaza-spiro[4.5]deca-enone template is prepared as described in Scheme 27.The α-bromination of the enone followed by palladium catalyzed couplingwith 4-amidinoboronic acid will give the desired alkylatedspiro-alkylated amidine. The reduction of tosylhydrazone of α,β-unsaturated ketones with sodium cyanoborohydride will give the alkenewith the double bond migration (see R. O. Hutchins, M. Kaucher, and L.Rua, J. Org. Chem, 1975, 40, 923). The deprotection with TFA followed byalkylation with appropriate halogenated alkylester or acylation withappropriate ester-acid chloride will give the desired N-alkylated orN-acylated intermediates. After mild basic hydrolysis of the ester willgive the desired amidino carboxylic acids.

The Scheme 34 describes the synthesis of8-benzoyl-2-amino-3-oxa-1,8-diaza-spiro[4.5]dec-1-enes in which thenitrogen in the 8-position is acylated. The N-Benzyl-4-piperidone wasconverted to the corresponding amino nitrile by the strecker reaction(see A. A. Cordi, J M. Lacoste, C. Courchay, P M. Vanhoutte, J. Med.Chem, 1995, 38, 4056). The stepwise hydrolysis of aminonitrile to aminoacid followed by reduction of acid to alcohol. The debenzylation iscarried out by catalytic hydrogenation and insitu protection withBoc-anhydride. The amino alcohol is cyclized with cyanogen bromide togive spiro-oxazoline ring. The 2-amino is acylated with appropriateester-acid chloride or alkylated with appropriate halogenatedalkylester. The protecting group is removed with trifluoroacetic acidfollowed by acylation with the 4-cyanobenzoyl chloride (or2-fluoro-4-cyanobenzoyl chloride) in presence of base such astriethylamine. After mild basic hydrolysis of ester, the nitrile isconverted to the amidine as described in earlier schemes, thus providingthe desired amidino carboxylic acids.

The Scheme 35 describes the synthesis of2-benzoyl-3-oxa-1,8-diaza-spiro[4.5]dec-1-ene in which the nitrogen inthe 2-position is benzoylated. The 2-amino spiro-oxazoline is acylatedwith 4-cyanobenzoyl chloride. The protecting group is removed withtrifluoroacetic acid followed by acylation with appropriate ester-acidchloride. After mild basic hydrolysis of ester, the nitrile is convertedto amidino to give the desired product.

The Scheme 36 describes the synthesis of2-benzoyl-3-oxa-1,8-diaza-spiro[4.5]dec-1-ene in which the nitrogen inthe 2-position is benzoylated. The 2-amino spiro-oxazoline is acylatedwith 4-cyanobenzoyl chloride. The protecting group is removed withtrifluoroacetic acid followed by alkylation with appropriate halogenatedalkylester. After mild basic hydrolysis of ester, the nitrile isconverted to amidino to give the desired product.

The Scheme 37 describes the synthesis of(8-benzoyl-aza-spiro[5.4]dec-3-yl)acetic acid derivatives. The reactionof N-benzyl-piperidone with diethyl lithiopyrrolidinomethyl phosphonatewill give enamine. The enamine can be directly alkylated with2-bromo-3-iodopropene, followed by aqueous hydrolysis to give thealkylated aldehyde. The hydrolysis of vinyl bromide can be readilyachieved with mercuric acetate and boron trifluoride etherate in glacialacetic acid at room temperature (see S. F. Martin, and T. Chou, J. Org.Chem. 1978, 43, 1027). The γ-keto aldehyde will be treated with aqueouspotassium hydroxide in methanol to afford cycloaldolization anddehydration to give the key intermediate spiro[4.5]dienone. The sidechain is introduced by Horner-Emmons reaction with triethylphosphonoacetate or triethyl phoshono propionate/sodium hydride. The bicyclicnucleus and the exo cyclic double bond are saturated and the protectivegroup is removed by catalytic hydrogenation with palladium hydroxide oncarbon. The subsequent acylation with 4-cyanobenzoyl chloride or(2-fluoro-4-cyanobenzoyl chloride), formation of amidine, and hydrolysisof the ester are carried out by methods described in the previousSchemes.

The Scheme 38 describes the synthesis8-benzoyl-1-3-acyl-spiro[4.5]dec-1-en. The mono protectedspiro-imidazoline from Scheme 33 is acylated with appropriate ester-acidchloride. The protecting group is removed followed with acylation with4-cyanobenzoyl chloride (or 2-fluoro-4-cyanobenzoyl chloride) inpresence of base such as triethylamine. After ester hyrolysis, thenitrile is converted to amidine funtionality as described in earlierschemes.

Scheme 40 describes the synthsis of a lactam. The imide was reduced withLiAIH₄ in THF to afford the amine. It was then N-debenzylated underhydrogenolysis conditions. The free piperidino compound was reacted withcarboxy group bearing compounds as described before. De-protection ofthe carboxy groups followed by conversion of the cyano to amidinoyielded the desired compounds.

Scheme 41 1-Benzylpiperidone was condensed with 4-methoxybenzylaminefollowed by reaction with potassium cyanide. The cyano group washydrolysed by reaction with hot conc. HCl. Then it was coupled with4-carboxyamido aniline to afford the amide. The 4-methoxy benzylprotective group was removed by reaction with DDQ, followed by reactionof the free amine with carbonyl diimidazole to give rise to ureaderivative. N-De-benzylation was achieved under hydrogenolysisconditions, followed by coupling with carboxy group bearing synthons.

Scheme 42 1-Benzylpiperidone was condensed with ethyl cyanoacetate andammonia in ethanol as described in the literature {S. M. McElvain, R. E.Lyle, Jr.; J. Amer. Chem. Soc. 72, 384 (1950)}. This was then treatedwith conc. Hcl at reflux for 3 days to afford the diacid, which waspurified by its conversion to diethylester followed by hydrolysis backto diacid. This was then converted to the anhydride, and treated with4-carboxyamido aniline as described in scheme 1 to afford the imide.This imide was converted to lactam by reaction with NaBH₄ as describedin Scheme 39. The rest of the sequence was carried out as describedelsewhere in this application.

Scheme 43 1-Benzylpiperidone was coupled with 4-carboxyamide aniline,and the imide was reacted with potassium cyanide. This amine nitrile wasreacted with potassium cyanate under acidic conditions to yield thehydantoin. The hydantoin functionality was reduced to urea by reactionwith NaBH₄, as described before.

Scheme 44 1-Benzylpiperidone was coupled with 4-methoxy benzylamine, andthe imine was then reacted with 4-carboxamide aniline to yield the aminenitrile. The 4-methoxy benzyl group was deprotected by reaction withDDQ. The free amine thus obtained was treated with potassium cyanateunder acidic conditions to afford the hydantoin. The rest of thesequence is described elsewhere in this patent.

Scheme 45 1-Benzylpiperidone was coupled with ethyl cyanoacetate toafford the addition product, which was then reacted with potassiumcyanide in ethanol/water at reflux to yield the dinitrile. It wasreduced with LiAIH4 to afford the diamine. It was then reacted withcarbonyl diimidazole to yield the 7-membered urea. This was subjected toN-debenzylation followed by coupling of the free piperidino compoundwith 4-cyano phenyl triflate under palladium catalysis. The rest of thesequence is described elsewhere.

Scheme 46 4-carboxamide aniline was reacted with the epoxide (synthesisof it is described elsewhere in this application) to afford the aminol.The hydroxy group was protected as tert-butyl dimethylsilyl ether, andthe amine was then reacted with chloro acetyl chloride. Then the TBSprotective group was removed and the free hydroxy compound was cyclizedto yield the spiro compound the rest of the steps are identical toprocedures described in other schemes

The following Examples illustrate the practice of the invention.

EXAMPLE 1 Preparation of Ethyl(3-(4-(Aminoiminomethyl)benzoyl)-3-aza-spiro[5.5]undec-9-yl)acetateHydrochloride

Step A: Preparation of benzyl9-oxo-3-aza-spiro[5.5]undec-7-ene-3-carboxylate

A solution of 0.98 g (3.96 mmol) benzyl 4-formylpiperidine-1-carboxylate(prepared by the method from Eur. J. Med. Chem. 1991, 26, 625) and 0.28g (4.0 mmol) 3-buten-2-one in a mixture of 1 ml methanol and 1 ml waterwas added dropwise to a boiling solution of 0.015 g (0.27 mmol)potassium hydroxide in 1 ml methanol. After 1 hour heating with refluxthe cooled mixture was poured into 50 ml water, and it was extractedwith tert.-butylmethylether. The organic layer was dried over sodiumsulfate and concentrated under reduced pressure. The title compound wasobtained from the remaining oil by HPLC on silica gel withhexane/acetone 4:1.

yield: 0.23 g (19%) oil

Step B: Preparation of benzyl9-((ethoxycarbonyl)methylene)-3-aza-spiro[5.5]undec-7-ene-3-carboxylate

23.63 g (105.4 mmol) diethyl ethoxycarbonylmethylphosphonate weredissolved in 500 ml dry THF, and the solution was kept under anatmosphere of nitrogen. After addition of 3.07 g (102.3 mmol) 80% sodiumhydride in mineral oil the mixture was stirred for 30 min at 0° C. Asolution of 30.0 g (100.2 mmol) of the Spiro compound from the previousstep in 33 ml dry THF was added dropwise, and it was stirred for 4 hourswith warming to room temperature. The mixture was poured into water, andit was extracted with tert.-butylmethylether. The organic layer waswashed two times with water and dried over sodium sulfate, and thesolvent was removed in vacuo. The title compound was purified bychromatography on silica gel with hexane/acetone 4:1 and obtained infive oily fractions each of them containing some impurities. They werecombined and used for the next step.

Total yield: 10.58 g (29%)

Step C: Preparation of ethyl (3-aza-spiro[5.5]undec-9-yl)acetate

9.6 g (26.0 mmol) of the protected Spiro compound from Step B weredissolved in 400 ml ethanol, and the solution was filled into ahydrogenation vessel followed by 2 g Pd(II) hydroxide on charcoal. Thereaction was carried out at 40° C. and 1 atm hydrogen, and it wascomplete after about 40 hours. The catalyst was removed by filtrationthrough silica gel, and the filtrate was concentrated under reducedpressure. The remaining oil was treated with dry ether, filtered, andthe filtrate was acidified with ethereal hydrochloric acid. Thehydrochloride of the title compound was collected by filtration, washedtwo times with ether, dissolved in water, and the solution was madealkaline with 2 N aqueous sodium hydroxide solution. After extractionwith dichloromethane the organic layer was dried over sodium sulfate andconcentrated in vacuo.

yield: 3.43 g (55%) oil

Step D: Preparation of ethyl(3-(4-cyanobenzoyl)-3-aza-spiro[5.5]undec-9-yl)acetate

0.24 g (1.0 mmol) of the compound from the previous step and 0.6 ml drypyridine were dissolved in 7 ml dry THF. The solution was kept below 5°C., while 0.165 g (1.0 mmol) 4-cyanobenzoyl chloride in 6 ml dry THFwere added dropwise within 15 min. After 2 hours stirring at roomtemperature it was concentrated under reduced pressure. The residue wastreated with water and extracted with ethyl acetate. The organic layerwas washed with diluted aqueous Cu(II) sulfate solution, dried oversodium sulfate, and the solvent was removed in vacuo. The title compoundwas obtained by HPLC on silica gel with hexane/acetone 4:1 followed by3:1.

yield: 0.19 g (51%) yellow oil

Step E: Preparation of ethyl(3-(4-(aminoiminomethyl)benzoyl)-3-aza-spiro[5.5]undec-9-yl)acetatehydrochloride

A solution of 1.65 g (4.48 mmol) of the nitrile from the previous stepin 75 ml ethanol was kept below 5° C. and saturated with gaseoushydrogen chloride. It was stirred over night at room temperature untilthe reaction was complete, and the solvent was removed under reducedpressure to give the crude intermediate imidoester. It was treated with50 ml 12% ethanolic ammonia solution and stirred for two days at roomtemperature. The mixture was concentrated in vacuo, and the residue wasstirred with 10 ml dichloromethane. Remaining solids were removed byfiltration, and the solvent was evaporated from the filtrate underreduced pressure. The crude title amidine was obtained by chromatographyon silica gel with dichloromethane containing between 5% and 10%ethanol. It solidified by stirring several times with ether, and it wasfurther purified by HPLC using the same eluent.

yield: 0.18 g (9.5%) colorless crystals, m.p.>300° C.

ELISA: IC₅₀>10 μM

PRP (ADP): IC₅₀=100 μM

EXAMPLE 2 Preparation of(3-(4-(Aminoiminomethyl)benzoyl)-3-aza-spiro[5.5]undec-9-yl)acetic AcidHydrochloride

70 mg (0.166 mmol) of the ester from Example 1 were treated with 2 ml 2N aqueous hydrogen chloride and 5 ml water. After heating for 4 hours at60° C. the solvent was removed under reduced pressure, and the remainingtitle acid was stirred with tert.-butylmethylether, collected byfiltration, and dried in vacuo.

yield: 64 mg (98%) beige hygroscopic solid

ELISA: IC₅₀=0.0082 μM

PRP (ADP): IC₅₀=0.41 μM

EXAMPLE 3 Preparation of Ethyl4-(3-(4-(Aminoiminomethyl)phenyl)-2-oxo-l-oxa-3,8-diaza-spiro[4.5]dec-8-yl)butanoateHydrochloride

Step A: Preparation of benzyl4-((N-(4-cyanophenyl)amino)methyl)-4-hydroxypiperidine-1-carboxylate

A mixture of 20.0 g (80.9 mmol) benzyl1-oxa-6-aza-spiro[2.5]octane-6-carboxylate (prepared from the protectedpiperidone and dimethylsulfoxonium methylide according to J. Med. Chem.1983, 26, 855; U.S. Pat. No. 4,353,901) and 47.0 g (397.8 mmol)4-aminobenzonitrile were heated at 160° C. for 30 hours until nostarting epoxide could be detected by TLC. It was cooled to roomtemperature, and the excess of the aniline was removed by chromatographyon silica gel with dichloromethane. The title compound was obtainedafter enhancing the polarity of the eluent by addition of 5% ethanol.

yield: 24.5 g (83%) beige crystalline solid, m.p.107-108° C.

Step B: Preparation of benzyl(3-(4-cyanophenyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-8-yl)carboxylate

A solution of 18.5 g (50.6 mmol) of the piperidine from Step A and 11.45g (70.6 mmol) N,Ni-carbonyldiimidazole in 210 ml dry THF were heatedwith reflux for 8 hours under an atmosphere of argon. After addition ofanother 5.0 g (30.8 mmol) carbonyldiimidazole heating was continuedovernight. The solvent was removed under reduced pressure, and the titlecompound was obtained by chromatography on silica gel withdichloromethane. It solidified from the combined pure fractions bystirring with hexane, was filtered, and dried in vacuo.

yield: 5.3 g (27%) pale yellow powder, m.p. 138-140° C.

Step C: Preparation of4-(2-oxo-l-oxa-3,8-diaza-spiro[4.5]dec-3-yl)benzonitriletrifluoroacetate

4.26 g (10.88 mmol) of the protected spiro compound from the previousstep were added in small portions to 26 ml trifluoroacetic acid, whilethe temperature was kept below 10° C. After two days stirring at roomtemperature the mixture was poured into ice water and neutralized withsodium bicarbonate. It was treated with dichloromethane, and aprecipitate of the title compound was formed between the two layers,which was collected by filtration and dried in vacuo.

yield: 4.0 g (99%) colorless crystals, m.p. 217-220° C.

Step D: Preparation of ethyl4-(3-(4-cyanophenyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-8-yl)butanoate

3.0 g (8.08 mmol) of the piperidine from the previous step weredissolved in 50 ml dry acetone followed by addition of 1.68 g (15.85mmol) sodium carbonate and 1.56 g (8.00 mmol) ethyl 4-bromobutanoate.The mixture was heated with reflux overnight, cooled to roomtemperature, and the inorganic solids were removed by filtration. Thefiltrate was concentrated under reduced pressure, and the title compoundwas obtained from the residue by chromatography on silica gel withdichloromethane/ethanol 40:1 and 20:1. The combined pure fractionssolidified, and the crystalline ester was dried in vacuo at 50° C.

yield: 2.4 g (81%) colorless crystals, m.p. 78-80° C.

Step E: Preparation of ethyl4-(3-(4-(ethoxycarbonimidoyl)phenyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-8-yl)butanoatehydrochloride

A suspension of 2.4 g (6.46 mmol) of the nitrile from Step D in 140 mldry ethanol was saturated with gaseous hydrogen chloride, while thetemperature was kept below 10° C. It was stirred overnight at roomtemperature, and the solvent was removed under reduced pressure. Thetitle intermediate crystallized by stirring with hexane. It was filteredwith suction and dried in vacuo.

yield: 2.2 g (75%) pale yellow powder, m.p. 195-199° C.

Step F: Preparation of ethyl4-(3-(4-(aminoiminomethyl)phenyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-8-yl)butanoatehydrochloride

2.2 g (4.85 mmol) of the intermediate from the previous step weresuspended in 130 ml saturated ethanolic ammonia solution. It was stirredovernight at room temperature, and after addition of another 30 mlammonia solution stirring was continued for another day. The solvent wasremoved under reduced pressure., and the title amidine was obtained bychromatography on silica gel with dichloromethane/ethanol 2:1. Itcrystallized by stirring with tert.-butylmethylether, was filtered withsuction, and dried in vacuo at 50° C.

yield=0.55 g (27%), white powder, m.p. 127-129° C.

ELISA: not tested

PRP (ADP): not tested

EXAMPLE 4 Preparation of4-(3-(4-(Aminoiminomethyl)phenyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-8-yl)butanoicAcid Bistrifluoroacetate

Step A: Preparation of tert.-butyl4-(3-(4-cyanophenyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-8-yl)butanoate

10.0 g (26.9 mmol) of the intermediate from Example 3, Step C weredissolved in 250 ml dry acetone followed by addition of 5.7 g (53.8mmol) sodium carbonate and 4.81 g (26.9 mmol) tert.-butyl4-chlorobutanoate (prepared according to Chem. Ber. 1965, 98, 2312). Itwas heated with reflux overnight. After addition of another 2.85 g (26.9mmol) sodium carbonate and 2.4 g (13.4 mmol) of the tert.-butyl esterheating was continued for another day. The inorganic solids were removedby filtration, and the filtrate was concentrated under reduced pressure.The title nitrile was obtained as an oil, which solidified upon standingafter chromatographic purification on silica gel withdichloromethane/ethanol 40:1 and 20:1.

yield: 1.87 g (17%) beige amorphous solid, m.p. 92-94° C.

Step B: Preparation of tert.-butyl4-(3-(4-((tert.-butoxycarbonylamino)iminomethyl)phenyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-8-yl)butanoate

A steady stream of gaseous hydrogen sulfide was passed through asolution of 1.8 g (4.5 mmol) of the nitrile from the previous step in67.5 ml dry pyridine and 7.2 ml triethylamine for about 1 hour. Afterstirring over night the conversion to the thioamide was complete. Themixture was poured into water and extracted three times with ethylacetate. The combined organic layers were dried over sodium sulfate, andconcentrated in vacuo. It was dissolved in 100 ml toluene, evaporatedagain under reduced pressure, and this procedure was repeated for twotimes to remove remaining pyridine.

The crude thioamide was suspended in 22.5 ml acetone followed bydropwise addition of 2.25 ml methyl iodide. It was stirred for 1 hour atroom temperature, while the mixture became a clear solution, andevaporated to dryness in vacuo. The residue was treated with 18 mlmethanol followed by 1.35 g (17.5 mmol) ammonium acetate, and themixture was heated for 3 h at 60° C. After evaporation under reducedpressure the remaining oil was stirred two times withtert.-butylmethylether to leave the crude crystalline amidine.

It was added to the amidine a mixture of 45 ml THF/water 1:1 followed by4.05 g (29.3 mmol) potassium carbonate and 4.5 g (20.6 mmol) Boc₂O.After 45 min. stirring at room temperature it was diluted with 50 mlwater and extracted with ethyl acetate. The organic layer was dried oversodium sulfate and concentrated under reduced pressure. The titlecompound was obtained by chromatography on silica gel withdichloromethane/ethanol 20:1 followed by another chromatographicpurification with dichloromethane/ethanol 40:1.

yield: 0.2 g (8.6%) yellow oil

Step C: Preparation of4-(3-(4-(aminoiminomethyl)phenyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-8-yl)butanoicacid bistrifluoroacetate

0.2 g (0.39 mmol) of the protected amidine from Step B were treated with2 ml trifluoroacetic acid, and the mixture was stirred at roomtemperature for 2 hours. It was evaporated to dryness in vacuo, and thesolid residue was stirred several times with ether and with hexane. Thetitle acid was collected by filtration and dried at 50° C. in vacuo.

yield: 0.12 g (53%), beige crystalline solid, m.p.122-125° C.

ELISA: not tested

PRP (ADP): IC₅₀=3 μM

EXAMPLE 5 Preparation of Ethyl(3-(4-(Aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)acetateHydrochloride

Step A: Preparation of tert.-butyl(3-(4-cyanophenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)carboxylate

4-Cyanobenzohydroximinoyl chloride was prepared by chlorination of4-cyanobenzaldehyde oxime with N-chlorosuccinimide according to theprocedure from J. Org. Chem. 1980, 45, 3916 and tert.-butyl4-methylenepiperidine-1-carboxylate according to the procedure from Int.Pat. Appl. WO 94/13696.

To a solution of 15.82 g (80.2 mmol) of the piperidine and 14.48 g (80.2mmol) of the hydroximinoyl chloride in 150 ml dry methanol was addeddropwise the same volume of a methanolic solution of 17.8 mltriethylamine, while the temperature was kept below 28° C. Afterstirring at room temperature for two days another 8.7 g (48.2 mmol) ofthe hydroximinoyl chloride and 10.7 ml triethylamine were added in threeequal portions, each after a period of 2 hours. It was stirred overnight, and the precipitate of the title compound was collected byfiltration and washed with methanol. The crystals were stirred withtert.-butylmethylether, filtered with suction, and dried in vacuo. Thecombined filtrates were poured into water and extracted three times withtert.-butylmethylether. The combined extracts were dried over sodiumsulfate, concentrated under reduced pressure to leave another crop ofthe title compound, which was washed with a small amount of methanol anddried in vacuo.

total yield: 26.0 g (95%) white powder, m.p. 169-171° C.

Step B: Preparation of4-(1-oxa-2,8-diaza-spiro[4.5]dec-2-en-3-yl)benzonitrile

10.0 g (29.3 mmol) of the protected piperidine from the previous Stepwere added in small portions to 25 ml trifluoroacetic acid, while thetemperature was maintained below 30° C. After 3 hours stirring at roomtemperature the mixture was carefully poured into saturated aqueoussodium bicarbonate solution and stirred for 20 min. A first crop of thetitle piperidine precipitated from the mixture and was collected byfiltration, dried in vacuo at 40° C., stirred with ethyl acetate, andfiltered again. The aqueous filtrate was adjusted to pH 11 with 2 Nsodium hydroxide solution and extracted four times with ethyl acetate.The combined organic layers were dried over sodium sulfate, and twoother crops were formed by gradual concentration under reduced pressure,which were washed with a small amount of ethyl acetate and filtered withsuction.

total yield: 6.94 g (98%) colorless crystals, m.p.>199° C. (dec.)

Step C: Preparation of ethyl(3-(4-cyanophenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)acetate

0.6 g (2.5 mmol) of the piperidine from Step B were dissolved in amixture of 10 ml dry DMF and 10 ml dry ethanol followed by addition of0.69 g (5.0 mmol) potassium carbonate. After 30 min stirring 0.42 g (2.5mmol) ethyl bromoacetate were added dropwise, and the mixture wasstirred overnight at room temperature. It was poured into water andextracted two times with ethyl acetate. The combined organic layers werewashed two times with water, dried over sodium sulfate, and concentratedunder reduced presure. The title compound was obtained from the residueby chromatography on silica gel with hexane/acetone 3:2 as an oil, whichsolidified upon standing.

yield: 0.52 g (64%) pale yellow crystals, m.p. 116-118° C.

Step D: Preparation of ethyl(3-(4-(aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)acetatehydrochloride

A solution of 0.5 g (1.53 mmol) of the nitrile from the previous Step in35 ml dry ethanol was saturated with gaseous hydrogen chloride at 0° C.The mixture was stirred overnight at room temperature, concentratedunder reduced pressure, and treated with 40 ml of a saturated ethanolicsolution of ammonia. After stirring over night another 10 ml of theammonia solution were added, and the reaction was brought to completionby standing at room temperature for another two days. The solvent wasremoved in vacuo, and the remaining solid title compound was purified bystirring with ethyl acetate, filtration, and chromatography on silicagel with dichloromethane/methanol 4:1.

yield: 0.23 g (40%), white powder, m.p.>254° C. (dec.)

ELISA: not tested

PRP (ADP): not tested

EXAMPLE 6 Preparation of(3-(4-(Aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)aceticAcid

0.12 g (0.315 mmol) of the ester from Example 5 were dissolved in 2 mlethanol, and after addition of 0.4 ml 2 N aqueous sodium hydroxidesolution the mixture was stirred overnight at room temperature. It wasadjusted to pH 4 with diluted acetic acid. The solvent was removed fromthe clear solution under reduced pressure to leave a brown oil, whichsolidified by stirring with a mixture of ethanol and water. It wasfiltered, stirred in a small amount of hot ethanol, filtered again, anddried in vacuo.

yield: 88 mg (88%) colorless amorphous solid, m.p.>265° C.

ELISA: not tested

PRP (ADP): IC₅₀=4 μM

EXAMPLE 7 Preparation of Ethyl4-(3-(4-(Aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)butanoateHydrochloride

Step A: Preparation of ethyl4-(3-(4-cyanophenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)butanoate

1.2 g (5.0 mmol) of the piperidine from Example 5, Step B, 1.37 g (9.9mmol) potassium carbonate, and a catalytic amount of potassium iodidewere suspended in 30 ml dry acetone followed by addition of 0.99 g (5.1mmol) ethyl 4-bromobutanoate. The mixture was heated with refluxovernight, cooled to room temperature, diluted with water, and extractedthree times with ethyl acetate. The combined organic layers were washedwith water, dried over sodium sulfate, and concentrated under reducedpressure. The title compound was purified by chromatography on silicagel with dichloromethane containing 5% ethanol and recrystallized fromether.

yield: 0.59 g (33%) white powder, m.p. 85-88° C.

Step B: Preparation of ethyl4-(3-(4-(aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)butanoatehydrochloride

0.58 g (1.63 mmol) of the nitrile from Step A were dissolved in 35 mldry ethanol and converted to the title amidine as described for Example5, Step D. It was purified by chromatography on silica gel withdichloromethane/methanol 7:3.

yield: 0.4 g (60%) colorless foam

ELISA: not tested

PRP (ADP): IC₅₀=2.8 μM

EXAMPLE 8 Preparation of4-(3-(4-(Aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)butanoicAcid

0.28 g (0.685 mmol) of the ethyl ester from Example 7 were hydrolized ina mixture of 4 ml ethanol and 0.9 ml 2 N aqueous sodium hydroxidesolution according to the procedure from Example 6. The title acidcrystallized from water/ethanol 1:2, and it was filtered and dried invacuo.

yield: 0.09 g (38%), colorless crystals, m.p.>185° C. (dec.)

ELISA: not tested

PRP (ADP) : IC₅₀=0.32 μM

EXAMPLE 9 Ethyl4-(3-(4-(Aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)-4-oxobutanoateHydrochloride

Step A: Preparation of4-(3-(4-cyanophenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)-4-oxobutanoicacid

To a suspension of 1.2 g (5.0 mmol) of the intermediate from Example 5,Step B in 30 ml dry THF were added 0.5 g (5.0 mmol) succinic anhydrideand 60 mg 4-N,N-dimethylaminopyridine (DMAP). After 30 min. heating withreflux another 0.15 g (1.5 mmol) succinic anhydride were added andrefluxing was continued from 1.5 hours. The solvent was removed underreduced pressure, and the title compound was obtained by chromatographyon silica gel with dichloromethane/ethanol 9:1. It was recrystallizedfrom ether, filtered, and dried in vacuo.

yield: 0.98 g (58%) white powder, m.p. 167-170° C.

Step B: Preparation of ethyl4-(3-(4-(aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)-4-oxobutanoatehydrochloride

A solution of 0.98 g (2.87 mmol) of the nitrile from Step A in 65 ml dryethanol was converted to the amidine with by saturation with hydrogenchloride followed by addition of 65 ml saturated ethanolic ammoniasolution as described for Example 5, Step D. It was purified bychromatography on silica gel with dichloromethane/ethanol 4:1.

yield: 0.48 g (40%), oil which solidified upon standing

ELISA: not tested

PRP (ADP): not tested

EXAMPLE 10 Preparation of4-(3-(4-(Aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)-4-oxobutanoicAcid

0.34 g (0.8 mmol) of the ethyl ester from Example 9 were hydrolized in amixture of 5 ml ethanol and 1 ml 2 N aqueous sodium hydroxide solutionaccording to the procedure from Example 6. A first crop (200 mg) of thetitle acid crystallized from water/ethanol 1:2. The mother liquid wasconcentrated, and a second crop (60 mg) was obtained by stirring with amixture of ethyl acetate and ether. The crystals were filtered and driedin vacuo.

total yield: 0.26 g (90%) white powder, m.p.>255° C. (dec.)

ELISA: not tested

PRP (ADP): IC₅₀=1 μm

EXAMPLE 11 Preparation of Ethyl4-(9-(4-(Aminoiminomethyl)benzoyl)-3-oxo-1-oxa-4,9-diaza-spiro[5.5]undec-4-yl)butanoateHydrochloride

Step A: Preparation of9-(4-cyanobenzoyl)-3-oxo-1-oxa-4,9-diaza-spiro[5.5]undecane

18.0 g (71.7 mmol) 3-oxo-1-oxa-4,9-diaza-spiro[5.5]undecane hydrobromide(prepared according to J. Med. Chem. 1983, 26, 855) were dissolved in300 ml dry pyridine, and 11.9 g (71.9 mmol) 4-cyanobenzoyl chloride wereadded in small portions. The mixture was stirred over night at roomtemperature and evaporated under reduced pressure. The residue wasdissolved in toluene, which was removed in vacuo. This process wasrepeated two times to remove remaining pyridine. It was treated withwater and neutralized by addition of saturated aqueous sodiumbicarbonate solution. The precipitate of the title compound was filteredwith suction, washed with water, and dried in vacuo.

yield: 13.6 g (63%) white powder, m.p. 250-251° C.

Step B: Preparation of ethyl4-(9-(4-cyanobenzoyl)-3-oxo-1-oxa-4,9-diaza-spiro[5.5]undec-4-yl)butanoate

A mixture of 2.99 g (10.0 mmol) of the compound from the previous stepand 100 ml dry DMF was warmed to 60° C. followed by addition of 0.44 g(11.0 mmol) sodium hydride (60% in mineral oil). It was kept withstirring for 30 min at this temperature until a clear solution wasobtained. 2.14 g (10.97 mmol) ethyl 4-bromobutanoate were added dropwiseafter cooling to room temperature. The mixture was stirred over night,concentrated to dryness under reduced pressure, and treated with waterand ethyl acetate. 1.7 g of remaining insoluble starting material wasrecovered by filtration. The organic layer was washed two times withbrine, dried over sodium sulfate, and concentrated in vacuo. The titleester was obtained by chromatography on silica gel with ethylacetate/ethanol 9:1.

yield: 0.8 g (19%) oil

Step C: Preparation of ethyl4-(9-(4-(aminoiminomethyl)benzoyl)-3-oxo-1-oxa-4,9-diaza-spiro[5.5]undec-4-yl)butanoatehydrochloride

A solution of 0.8 g (1.93 mmol) of the nitrile from the previous step in50 ml dry ethanol was kept at 10° C. and saturated with gaseous hydrogenchloride. It was stirred over night at room temperature and concentratedto dryness under reduced pressure to leave the crude intermediateiminoester, which was treated with 50 ml 15% ethanolic ammonia andstirred again over night. The solvent was removed in vacuo, and thetitle amidine was obtained by chromatography on silica gel withethanol/ethyl acetate 7:3.

yield: 0.11 g (12%) colorless foam

ELISA: not tested

PRP (ADP): not tested

EXAMPLE 12 Preparation of4-(9-(4-(Aminoiminomethyl)benzoyl)-3-oxo-1-oxa-4,9-diaza-spiro[5.5]undec-4-yl)butanoicAcid Hydrochloride

To a solution of 75 mg (0.16 mmol) of the ester from Example 11 in 10 mlethanol was added 1 ml 2 N aqueous sodium hydroxide solution, and themixture was stirred over night at room temperature. It was concentratedto dryness in vacuo, and the residue was dissolved in a small amount ofwater. It was acidified with 1 ml aqueous 2 N hydrochloric acid and thesolvent was removed under reduced pressure. The residue was stirredseveral times with ethanol. The combined extracts were filtered througha plug of cotton wool and concentrated to dryness in vacuo to leave thepure title acid.

yield: 58 mg (82%) colorless foam

ELISA: not tested

PRP (ADP): IC₅₀>10 μM

EXAMPLE 13 Preparation of(8-(4-(Aminoiminomethyl)benzoyl)-1-phenylmethyl-2,4-dioxo-1,3,8-triaza-spiro[4.5]dec-3-yl)aceticAcid Trifluoroacetate

Step A: Preparation of benzyl4-benzylamino-4-cyanopiperidine-1-carboxylate hydrochloride

11.65 g (50 mmol) N-(benzyloxycarbonyl)piperidone (prepared from4-piperidone and benzyl chloroformate according to Chem. Pharm. Bull.1982, 30, 1084) and 7.2 g (50 mmol) benzylamine hydrochloride weredissolved in a mixture of 20 ml methanol and 10 ml water. The solutionwas kept at 0° C., while 15 ml of an aqueous solution of 3.26 g (50mmol) potassium cyanide were added dropwise. After stirring overnight atroom temperature a crystalline solid had been formed, which wascollected by filtration. The filtrate was treated with a mixture ofwater and ether. The organic layer was separated and the aqueous layerextracted with ether. The combined organic layers were dried over sodiumsulfate and added to the solid material. The title hydrochlorideprecipitated after treating with a saturated ethereal solution ofhydrogen chloride, and it was isolated by filtration and dried in vacuo.

yield: 18.25 g (95%) white powder, m.p. 143-145° C. (dec.)

Step B: Preparation of benzyl1-enzyl-2,4-dioxo-1,3,8-triaza-spiro[4.5]decane-8-carboxylate

To a solution of 17.3 g (44.8 mmol) of the piperidine from the previousStep in 30 ml acetic acid were added 15 ml of an aqueous solution of 7.3g (90.0 mmol) potassium cyanate. The mixture became a clear solutionwith slight warming. It was stirred for 30 min at room temperaturefollowed by 45 min at 55° C. and poured into ice water. The aqueoussolution was extracted two times with ethyl acetate, and the organiclayer was concentrated in vacuo. The residue was treated with 45 ml 10%aqueous hydrochloric acid. It was kept at 55° C. for 15 min and dilutedwith water. After two extractions with ethyl acetate the combinedextracts were washed with aqueous sodium bicarbonate solution, driedover sodium sulfate, and concentrated in vacuo. The remaining oil of thepure title hydantion slowly solidified upon standing.

yield: 13.25 g (75%) colorless amorphous solid, m.p.123-125° C.

Step C: Preparation of1-phenylmethyl-2,4-dioxo-1,3,8-triaza-spiro[4.5]decane

8.0 g (20.3 mmol) of the protected piperidine from Step C were dissolvedin 100 ml ethanol. After addition of 500 mg 10% Pd/C the mixture wasfilled into an autoclave and hydrogenated for 32 hours at 50 atmhydrogen and 50° C. The catalyst was removed by filtration, and thesolution was concentrated under reduced pressure. The title compound wasobtained from the residue by chromatography on silica gel withdichloromethane containing from 5% to 20% ethanol.

yield: 1.8 g (34%) pale yellow amorphous solid, m.p. 195-196° C.

Step D: Preparation of8-(4-cyanobenzoyl)-1-phenylmethyl-2,4-dioxo-1,3,8-triaza-spiro[4.5]decane

1.7 g (6.56 mmol) of the piperidine from the previous Step weredissolved in a mixture of 50 ml dry THF and 8 ml dry pyridine. Aftercooling with ice 1.1 g (6.64 mmol) 4-cyanobenzoyl chloride were added.It was stirred overnight at room temperature and poured into ice water.After two extractions with ethyl acetate the combined organic layerswere successively washed two times with saturated aqueous Cu(II) sulfatesolution and with brine. It was dried over sodium sulfate andconcentrated under reduced pressure. The title compound was isolatedfrom the residue by chromatography on silica gel with dichloromethanecontaining 4% ethanol.

yield: 0.92 g (36%) colorless crystals, m.p. 207-209° C.

Step E: Preparation of tert.-butyl(8-(4-cyanobenzoyl)-1-phenylmethyl-2,4-dioxo-1,3,8-triaza-spiro[4.5]dec-3-yl)acetate

0.9 g (2.3 mmol) of the compound from the previous Step were dissolvedin 30 ml dry THF. After addition of 55 mg (2.3 mmol) sodium hydride itwas stirred for 30 min at room temperature followed by addition of 0.585g (3.0 mmol) tert.-butyl bromoacetate. The mixture was stirred for twodays and poured into ice water. It was extracted two times with ethylacetate. The combined organic layers were dried over sodium sulfate andconcentrated in vacuo, and the title hydantion was obtained bychromatography on silica gel with dichloromethane/ethanol 96:4.

yield: 1.12 g (96%) oil

Step F: Preparation of tert.-butyl(8-(4-((tert.-butoxycarbonylamino)iminomethyl)benzoyl)-1-phenylmethyl-2,4-dioxo-1,3,8-triaza-spiro[4.5]dec-3-yl)acetate

0.5 g (1.0 mmol) of the nitrile from Step E were dissolved in a mixtureof 15 ml dry pyridine and 1.6 ml dry triethylamine. It was saturatedwith gaseous hydrogen sulfide, left at room temperature overnight, andpoured into water. After three extractions with ethyl acetate thecombined organic layers were dried over sodium sulfate and concentratedunder reduced pressure. The residue was dissolved in 100 ml toluene,which was distilled off again in vacuo to remove traces of pyridine. Tothe remaining substance were added 5 ml acetone and 5 ml methyl iodide,and it was heated with reflux for 45 min, while a crystallineprecipitate was formed. It was concentrated under reduced pressure,dissolved in 4 ml dry methanol, treated with 0.3 g (3.9 mmol) ammoniumacetate and heated for 2 h at 60° C. The solvent was removed in vacuo,and the residue was stirred with ether. The solution was separated fromthe insoluble amidine, which was Boc-protected by dissolving in 10 ml ofa 1:1 mixture of water and THF followed by addition of 0.9 g (6.5 mmol)potassium carbonate and 1.0 g (4.6 mmol) Boc2O and stirring for 1 hourat room temperature. It was diluted with water and extracted with ethylacetate. The organic layer was dried over sodium sulfate andconcentrated under reduced pressure, and the title compound was isolatedby chromatography on silica gel with dichloromethane/ethanol 96:4.

yield: 0.24 g (39%) oil

Step G: Preparation of(8-(4-(aminoiminomethyl)benzoyl)-1-phenylmethyl-2,4-dioxo-1,3,8-triaza-spiro[4.5]dec-3-yl)aceticacid trifluoroacetate

220 mg (0.355 mmol) of the protected amidine from the previous Step werestirred at room temperature with 2 ml trifluoroacetic acid. After 2hours the solvent was removed in vacuo, and the residue was dissolved inwater. Again it was concentrated under reduced pressure. The title acidslowly precipitated by stirring with a mixture of ether and ethanol. Itwas collected by filtration and dried in vacuo.

yield: 0.05 g (24%) colorless powder, m.p.>197° C. (dec.)

ELISA: not tested

PRP (ADP): IC₅₀>100 μM

EXAMPLE 14 Preparation of Ethyl3-(3-(4-(Aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)propanoateHydrochloride

Step A: Preparation of methyl(3-(4-cyanophenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)propanoate

To a suspension of 2.0 g (8.3 mmol) of the intermediate from Example 5,Step B in 30 ml dry ethanol were added 0.715 g (8.3 mmol) methylacrylate. The mixture was stirred over night at room temperaturefollowed by 9 hours at 50° C. It was diluted with water and extractedfive times with ethyl acetate. The combined organic layers were driedover sodium sulfate and concentrated under reduced pressure. One crop(0.64 g) of the title compound was obtained after chromatography onsilica gel with ethyl acetate/ethanol 9:1 followed by recrystallizationfrom ethanol. Another crop (0.23 g) was obtained by concentration of theaqueous layer to a small volume, filtration of the precipitate, stirringin hot ethanol, another filtration, and drying in vacuo.

total yield: 0.87 g (32%) colorless crystals, m.p.>225° C. (dec.)

Step B: Preparation of ethyl3-(3-(4-(aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)propanoatehydrochloride

A solution of 0.39 g (1.19 mmol) of the nitrile from Step A in 30 ml dryethanol was saturated with gaseous hydrogen chloride followed bytreatment with 40 ml saturated ethanolic ammonia solution according tothe procedure from Example 5, Step D. The title amidine was obtained bychromatography on silica gel with dichloromethane/methanol 3:1.

yield: 0.23 g (49%), pale yellow crystals, m.p.>192° C. (dec.)

ELISA: not tested

PRP (ADP): IC₅₀=0.64 μM

EXAMPLE 15 Preparation of4-(3-(4-(Aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)propanoicAcid

0.26 g (0.66 mmol) of the ester from Example 14 were hydrolized in amixture of 4 ml ethanol and 0.9 ml 2 N aqueous sodium hydroxide solutionas described in Example 6.The title acid crystallized from 1.5 mlethanol/water 2:1.

yield: 0.1 g (46%) white powder, m.p.>243° C. (dec.)

ELISA: not tested

PRP (ADP): IC₅₀=0.11 μM

EXAMPLE 16 Preparation of Ethyl 5- (3- (4- (Aminoiminomethyl)phenyl)-1-oxa-2, 8-diaza-spiro[4.5]dec-2-en-8-yl) -5-oxopentanoateHydrochloride

Step A: Preparation of5-(3-(4-cyanophenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)-5-oxopentanoicacid

To a suspension of 1.5 g (6.22 mmol) of the intermediate from Example 5Step B in 40 ml dry THF were added 0.71 g (6.22 mmol) glutaric anhydrideand 76 mg 4-N,N-dimethylaminopyridine (DMAP). It was stirred for 1 h atroom temperature followed by 2 h heating with reflux. The solvent wasremoved under reduced pressure, and the title compound was obtained bychromatography on silica gel with dichloromethane containing 4% ethanol.It was recrystallized from ethyl acetate/ether, and the crystals werecollected by filteration and dried in vacuo.

yield: 0.97 g (44%) white powder, m.p. 183-185° C.

Step B: Preparation of ethyl5-(3-(4-(aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)-5-oxopentanoatehydrochloride

A suspension of 0.91 g (2.56 mmol) of the nitrile from the previous Stepin 60 ml dry ethanol was saturated with gaseous hydrogen chloridefollowed by treatment with 58 ml of a saturated ethanolic ammoniasolution according to the procedure from Example 5, Step D. The mixturewas concentrated under reduced pressure. A precipitate was removed byfiltration after treatment with dichloromethane/ethanol 4:1, and thepure title amidine was obtained by chromatography on silica gel usingthe same solvent.

yield: 0.49 g (44%), pale yellow oil, which solidified upon standing

ELISA: not tested

PRP (ADP): IC₅₀=0.2 μM

EXAMPLE 17 Preparation of5-(3-(4-(Aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)-5-oxopentanoicAcid

0.34 g (0.78 mmol) of the ethyl ester from Example 16 were dissolved in5 ml ethanol, and after addition of 1 ml 2 N aqueous sodium hydroxidesolution the mixture was stirred over night. It was brought to pH 5 withdiluted acetic acid, stirred for 30 min, and the title acid was filteredwith suction, washed with water and ethanol, successively, and dried invacuo at 50° C.

yield: 0.28 g (97%), colorless crystals, m.p.>280° C. (dec.)

ELISA: not tested

PRP (ADP): IC₅₀=0.084 μM

EXAMPLE 18 Preparation of Ethyl5-(3-(4-(Aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)pentanoateHydrochloride

Step A: Preparation of ethyl5-(3-(4-cyanophenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)pentanoate

To a suspension of 1.1 g (4.56 mmol) of the intermediate from Example 5,Step B in 20 ml dry ethanol were added 1.26 g (9.12 mmol) potassiumcarbonate. After 15 min stirring at room temperature 1.03 g (4.93 mmol)ethyl 5-bromopentanoate and a catalytic amount of potassium iodide wereadded. It was stirred over night at room temperature, and the reactionwas brought to completion by addition of another 0.52 g (2.49 mmol)ethyl 5-bromopentanoate and heating with reflux for 3 h. The mixture waspoured into water and extracted three times with ethyl acetate. Thecombined organic layers were washed with water, dried over sodiumsulfate, and concentrated under reduced pressure. The title compound wasobtained by chromatography on silica gel with dichloromethane/ethanol95:5 followed by 92:8.

yield: 0.57 g (34%), yellow oil, which solidified upon standing

Step B: Preparation of ethyl5-(3-(4-(aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)pentanoatehydrochloride

A solution of 0.55 g (1.49 mmol) of the nitrile from the previous stepin 35 ml dry ethanol was saturated with gaseous hydrogen chloride andconverted to the title amidine with 45 ml saturated ethanolic ammoniasolution as described in Example 9, Step B. It was purified bychromatography on silica gel with dichloromethane/ethanol 4:1 followedby 3:1, and it solidified after addition of some drops of ethanolicammonia.

yield: 0.51 g (81%), pale yellow amorphous solid, m.p. 146-148° C.

ELISA: not tested

PRP (ADP): not tested

EXAMPLE 19 Preparation of5-(3-(4-(Aminoiminomethyl)phenyl)-1-oxa-2,8-diaza-spiro[4.5]dec-2-en-8-yl)pentanoicAcid

To a solution of 0.4 g (0.946 mmol) of the ester from Example 18 in 6 mlethanol were added 1.2 ml 2 N aqueous sodium hydroxide solution. Themixture was stirred at room temperature over night, and stirring wascontinued for another 30 min after addition of another 0.2 ml sodiumhydroxide. It was brought to pH 5 with diluted acetic acid, and theprecipitate of the title compound was filtered with suction. Afterstirring with a small amount of water for 30 min, it was filtered again,washed with water and with ethanol, and dried in vacuo at 60° C.

yield: 0.125 g (37%) colorless amorphous solid, m.p. 236-238° C. (dec.)

ELISA: not tested

PRP (ADP): IC₅₀=0.45 μM

EXAMPLE 20 Preparation of(9-(2-(Pyridin-4-yl)ethyl)-3,9-diazaspiro[5.5]undec-3-yl)acetic AcidBistrifluoroacetate

Step A: Preparation of1,5-dicyano-2,4-dioxo-9-phenylmethyl-3,9-diazaspiro[5.5]undecane

A mixture of 159.7 g (0.844 mol) N-benzylpiperidone and 191.0 g (1.689mol) ethyl cyanoacetate was dissolved in a 12% ethanolic solution ofammonia, and it was kept in a refrigerator for 8 days, while the titlecompound precipitated from the solution. It was collected by filtration,washed with ethanol and ether, successively, and suspended in 910 mlwater. It was brought to pH 6 with 2N hydrochloric acid, stirred for 1h, filtered again, washed with water, and dried in vacuo.

yield: 177.2 g (65%) pale yellow crystals, m.p. 179-181° C.

Step B: Preparation of (4-(hydroxycarbonylmethyl)-1-phenylmethylpiperidin-4-yl)acetic acid hydrochloride

88.9 g (275.8 mmol) of the intermediate from the previous step wereheated in 550 ml 18% hydrochloric acid for 24 h. After addition of 100ml conc. HCl heating was continued for the same period. Solids wereremoved by filtration, and the title diacid precipitated uponconcentration of the filtrate to 60% of its volume under reducedpressure. It was filtered with suction, washed with ethanol, and driedin vacuo.

yield: 46.0 g (51%) white powder, m.p. 223-225° C.

Step C Preparation of9-phenylmethyl-3,9-diazaspiro[5.5]undecan-2,4-dione

11.6 g (35.4 mmol) of the diacid from Step B and 3.0 g (50.0 mmol) ureawere thoroughly mixed in a mortar, and it was heated at 160° C. for 6 h.After 30 minutes no gas evolution could be observed. It was cooled toroom temperature and stirred with ethanol. The solids were collected byfiltration, suspended in saturated aqueous sodium bicarbonate solution,and stirred until gas evolution had ended. The title compound wasfiltered with suction, washed with water, and dried in vacuo.

yield: 6.05 g (63%) colorless needles, m.p.209-210° C.

Step D Preparation of 3-phenylmethyl-3,9-diazaspiro[5.5]undecane

Under an atmosphere of argon was added in small portions to a suspensionof 8.4 g (221.3 mmol) LiAlH4 in 200 ml dry THF a suspension of 16.9 g(62.1 mmol) of the intermediate from the previous step in the samevolume of THF, while the temperature was kept between 0° C. and 10° C.It was stirred for 1 h at room temperature and heated with reflux foranother 4 h. The mixture was cooled and quenched with water. Solids wereremoved by filtration through a pad of silica gel, washed with ethanol,and the filtrate was concentrated under reduced pressure. The residuewas stirred with ether, and it was filtered again. The pure titleintermediate was obtained from the filtrate by evaporation of thesolvent in vacuo.

yield: 11.7 g (77%) pale yellow amorphous solid, m.p. 105-106° C. (dec.)

Step E Preparation of tert.-butyl(9-phenylmethyl-3,9-diazaspiro[5.5]undec-3-yl)acetate

A mixture of 10.0 g (40.9 mmol) of the compound from the previous step,8.55 g (43.83 mmol) tert.-butyl bromoacetate, and 6.2 g (44.9 mmol) drypotassium carbonate in 200 ml dry ethanol was heated with reflux for 3h. After cooling to room temperature solids were removed by filtrationand washed with ethanol. The filtrate was concentrated under reducedpressure, and the residue was stirred with tert.-butylmethylether. Itwas filtered again and washed with the same solvent. The filtrate waswashed four times with water, dried over sodium sulfate, andconcentrated in vacuo to leave the pure title compound.

yield: 9.37 g (64%) yellow oil

Step F Preparation of tert.-butyl (3,9-diazaspiro[5.5]undec-3-yl)acetate

To a solution of 9.3 g (25.9 mmol) of the compound from Step E in 400 mldry ethanol were added 1.5 ml acetic acid and 0.7 g Pd(II) hydroxide.The mixture was filled into an autoclave and hydrogenated at 50 atm and50° C. for 18 h. The solvent was removed under reduced pressure, and theresidue was stirred with saturated aqueous sodium bicarbonate solution.It was extracted five times with 100 ml tert.-butylmethylether followedby three extractions with 100 ml dichloromethane. The extracts weredried over sodium sulfate and concentrated in vacuo, separately. Thefirst extract contained a mixture of compounds, whereas the pure titleintermediate was obtaind from the following.

yield: 5.52 g (79%) oil, which solidified upon standing

Step G Preparation of tert.-butyl(9-(2-(pyridin-4-yl)ethyl)-3,9-diazaspiro[5.5]undec-3-yl)acetate

A solution of 2.5 g (9.3 mmol) of the compound from Step F and 0.98 g(9.3 mmol) 4-vinylpyridine in 20 ml dry acetonitrile was heated withreflux over night. The solvent was removed under reduced pressure, andthe title compound was obtained from the residue by chromatography onaluminum oxide with dichloromethane followed by dichloromethane/ethanol9:1.

yield: 2.49 g (72%) brown resin

Step H Preparation of(9-(2-(pyridin-4-yl)ethyl)-3,9-diazaspiro[5.5]undec-3-yl)acetic acidbistrifluoroacetate

0.1 g (0.268 mmol) of the ester from Step G in 2 ml trifluoroacetic acidwere stirred at room temperature for 30 minutes. The solvent was removedunder reduced pressure, and the remaining title compound was stirredwith ether, collected by filtration, and dried in vacuo.

yield: 0.12 g (82%) beige hygroskopic crystalline solid

ELISA: not tested

PRP (ADP): IC₅₀>100 μM

EXAMPLE 21 Preparation of4-(8-(4-(Aminoiminomethyl)benzoyl)-2,4-dioxo-1,3,8-triaza-spiro[4.5]dec-3-yl)butanoicAcid Trifluoroacetate

Step A Preparation of 1,3,8-triaza-spiro[4.5]decan-2,4-dione

To a solution of 14.6 g (48.1 mmol) benzyl2,4-dioxo-1,3,8-triaza-spiro[4.5]decan-8-carboxylate (prepared fromN-(benzyloxycarbonyl)piperidone by the method from J. Med. Chem. 1995,38, 3772) in 250 ml dry ethanol were added 500 mg 10% Pd/C, and themixture was filled into an autoclave. After stirring at 60° C. and 10atm of hydrogen for 3 h no starting hydantoin could be detected by TLC.A precipitate had been formed which was redissolved by addition of 100ml acetic acid. The catalyst was removed by filtration and the solutionwas concentrated in vacuo. Aqueous sodium bicarbonate solution was addeduntil the mixture became a clear solution, and the title compoundprecipitated in two crops upon concentration in vacuo.

total yield: 8.1 g (99%), white powder, m.p. 303-304° C. (dec.)

Step B: Preparation of8-(4-cyanobenzoyl)-1,3,8-triaza-spiro[4.5]decan-2,4-dione

7.6 g (45.9 mmol) 4-cyanobenzoyl chloride were added to a cooledsolution of 7.8 g (46.1 mmol) of the compound from the previous step ina mixture of 400 ml dry THF and 67 ml dry pyridine. After stirring overnight at room temperature the mixture was poured into ice-cold water. Itwas extracted two times with ethyl acetate, and the combined organiclayers were washed with diluted aqueous Cu(II) sulfate solution and withbrine, successively. It was dried over sodium sulfate, and the titlecompound precipitated upon concentration under reduced pressure. It wascollected by filtration and dried in vacuo.

yield: 4.9 g (36%), yellow crystalline solid, m.p. 144-146° C.

Step C Preparation of tert.-butyl4-(8-(4-cyanobenzoyl)-2,4-dioxo-1,3,8-triaza-spiro[4.5]dec-3-yl)butanoate

A mixture of 2.4 g (8.0 mmol) of the compound froms Step B, 1.8 g (10.1mmol) tert.-butyl 4-chlorobutanoate (prepared according to Chem. Ber.1965, 98, 2312), 2.8 g (20.3 mmol) dry potassium carbonate, and acatalytic amount potassium iodide in 50 ml dry DMF was heated at 100° C.for 6 h until the conversion was complete. It was poured into ice-coldwater, and the crude solid title compound was collected by filtration.It was redissolved in hot ethyl acetate and dried over sodium sulfate,which was removed by filtration. The title hydantoin crystallized fromthe filtrate by addition of hexane.

yield: 2.4 g (68%), colorless crystals, m.p. 225-226° C. (dec.)

Step D Preparation of tert.-butyl4-(8-(4-((tert.-butoxycarbonylamino)iminomethyl)benzoyl)-2,4-dioxo-,1,3,8-triaza-spiro[4.5]dec-3-yl)butanoate

A solution of 0.9 g (2.0 mmol) of the compound from Step C in a mixtureof 13 ml dry pyridine and 1.8 ml triethylamine was saturated withhydrogen sulfide during 2 h. After stirring over night at roomtemperature it was poured into water and extracted three times withethyl acetate. The combined organic layers were dried over sodiumsulfate and concentrated under reduced pressure followed by solution in100 ml toluene. The solvent was evaporated in vacua again, in order toremove remaining traces of pyridine. The residue was dissolved in 5 mlacetone, treated with 5 ml iodomethane, and heated with reflux for 75minutes. It was concentrated to dryness, dissolved in 5 ml methanol,treated with 0.3 g (3.9 mmol) ammonium acetate, and heated for 2 h at60° C. The solvent was removed in vacuo, and the solid residue waswashed by stirring with ether, dissolved in 20 ml of a mixture ofTHF/water 1:1 followed by addition of 1.8 g (13.0 mmol) potassiumcarbonate and 2.0 g (9.2 mmol) Boc₂O. After stirring over night at roomtemperature it was diluted with water, extracted with ethyl acetate, andthe organic layer was dried over sodium sulfate and concentrated underreduced pressure. The title compound was obtained from the residue bychromatography on silica gel with dichloromethane/ethanol 9:1.

yield: 0.41 g (36%), white powder, m.p. 133-135° C. (dec.)

Step E Preparation of4-(8-(4-(aminoiminomethyl)benzoyl)-2,4-dioxo-1,3,8-triaza-spiro[4.5]dec-3-yl)butanoicacid trifluoroacetate

0.19 g (0.34 mmol) of the protected amidine from the previous step werestirred in 1 ml trifluoroacetic acid at room temperature for 45 minutes.The mixture was concentrated to dryness under reduced pressure, and theresidue of the title compound crystallized by stirring in a mixture ofether and ethanol. It was filtered with suction and dried in vacuo.

yield: 0.14 g (80%), white powder, m.p. 226° C. (dec.)

ELISA: not tested

PRP (ADP): IC₅₀>10 μM

EXAMPLE 22 Ethyl(3-(2-(Pyridin-4-yl)ethyl)-3-azaspiro[5.5]undec-9-yl)acetate

A solution of 0.48 g (2.0 mmol) of the intermediate from Example 1, StepC and 0.21 g (2.0 mmol) 4-vinylpyridine in 6 ml acetonitrile were heatedat 80° C. over night. The solvent was removed under reduced pressure,and the remaining solid title compound was purified by stirring severaltimes with tert.-butylmethylether, filtered with suction, and dried invacuo.

yield: 0.17 g (25%), brown crystalline solid, m.p. 142° C.

ELISA: not tested

PRP (ADP): IC₅₀>100 μM

EXAMPLE 23 (3-(2-(Pyridin-4-yl)ethyl)-3-azaspiro[5.5]undec-9-yl)aceticAcid

To a solution of 0.05 g (0.145 mmol) of the ester from Example 22 in 2ml ethanol were added three drops of 2N aqueous sodium hydroxidesolution. The mixture was heated at 70° C. for 3 h and diluted with 10ml water. It was washed two times with dichloromethane, and the aqueouslayer was adjusted to pH 7-8 with hydrochloric acid. After anotherextraction with dichloromethane the aqueous layer was concentrated underreduced pressure and the residue stirred with 20 ml of a mixture ofethanol/dichloromethane 9:1. Solids were removed by filtration, and thefiltrate was concentrated in vacuo to leave the pure title acid.

yield: 0.2 g (44%), pale yellow resin

ELISA: not tested

PRP (ADP): IC₅₀>100 μM

EXAMPLE 24 Ethyl(3-(2-(Piperidin-4-yl)ethyl)-3-azaspiro[5.5]undec-9-yl)acetate

0.17 g (0.5 mmol) of the compound from Example 22 were dissolved in 25ml 90% acetic acid followed by addition of 30 mg platinum(IV) oxide. Themixture was filled into an autoclave and hydrogenated for 4 h at roomtemperature and a pressure of 20 atm. The catalyst was removed byfiltration and the solvent evaporated under reduced pressure. Theresidue was treated with water and neutralized with sodium carbonatefollowed by extraction with dichloromethane. The organic layer was driedover sodium sulfate and concentrated in vacuo to leave the pure titlecompound.

yield: 0.04 g (23%), brown resin

ELISA: not tested

PRP (ADP): IC₅₀>100 μM

EXAMPLE 25 (3-(2-(Piperidin-4-yl)ethyl)-3-azaspiro[5.5]undec-9-yl)aceticAcid

0.35 g (1.0 mmol) of the compound froms Example 24 were hydrolized in 15ml ethanol containing 7 drops aqueous sodium hydroxide as described forExample 23.The title acid solidified upon stirring in dichloromethane.It was filtered and dried in vacuo.

yield =0.03 g (9%), beige crystalline solid, m.p. 263-265° C.

ELISA: not tested

PRP (ADP): IC₅₀>10 μM

EXAMPLE 26(9-(2-(Pyridin-4-yl)ethyl)-2,4-dioxo-3,9-diazaspiro[5.5]undec-3-yl)aceticAcid Trifluoroacetate

Step A Preparation of tert.-butyl(9-phenylmethyl-2,4-dioxo-3,9-diazaspiro[5.5]undec-3-yl)acetate

To a suspension of 10.0 g (36.7 mmol) of the imid from Example 20, StepC in 100 ml dry DMF were added in small portions 0.9 g (37.5 mmol)sodium hydride, which had been purified from mineral oil by washing withhexane, and the mixture was stirred for 30 minutes at 50° C. Afteraddition of 7.0 g (35.9 mmol) tert.-butyl bromoacetate it was stirred atroom temperature over night, poured into 300 ml water, and extracted twotimes with tert.-butylmethylether. The combined organic layers weredried over sodium sulfate, concentrated in vacuo, and the title esterwas obtained by chromatography on silica gel with dichloromethanefollowed by dichloromethane/ethanol 96:4.

yield: 13.5 g (97%) oil, which solidified upon standing

Step B: Preparation of tert.-butyl(2,4-dioxo-3,9-diazaspiro[5.5]undec-3-yl)acetate

To a solution of 13.4 g (34.7 mmol) of the compound from Step A in 500ml ethanol were added 2 ml acetic acid and 0.9 g Pd(II) hydroxide. Themixture was filled into an autoclave and hydrogenated at 50° C. and 50atm for 20 h. The solvent was removed in vacuo, and the residue wasstirred with saturated aqueous sodium bicarbonate solution. It wasextracted several times with tert.-butylmethylether, and the combinedextracts were dried over sodium sulfate and concentrated under reducedpressure. The title compound was obtained by chromatography on silicagel with dichloromethane followed by dichloromethane/ethanol 7:3.

yield: 6.8 g (66%) oil, which solidified upon standing

Step C Preparation of tert.-butyl(9-(2-(pyridin-4-yl)ethyl)-2,4-dioxo-3,9-diazaspiro[5.5]undec-3-yl)acetate

A solution of 3.0 g (10.1 mmol) of the compound from the previous stepand 1.05 g (10.0 mol) 4-vinylpyridine in 20 ml dry acetonitrile wereheated with reflux for 6 h and stirred over night at room temperature.The solvent was removed under reduced pressure, and the title pyridinewas obtained by chromatography on silica gel with dichloromethanefollowed by dichloromethane/ethanol 9:1.

yield: 2.25 g (56%) beige resinous crystals

Step D Preparation of(9-(2-(pyridin-4-yl)ethyl)-2,4-dioxo-3,9-diazaspiro[5.5]undec-3-yl)aceticacid trifluoroacetate

0.1 g (0.25 mmol) of the ester from Step C were stirred in 2 mltrifluoroacetic acid at room temperature for 30 minutes. The solvent wasremoved in vacuo, and the remaining title compound was purified bytreating with ethanol and dichloromethane, successively, which wasremoved under reduced pressure.

yield: 0.11 g (96%), beige resinous crystals

ELISA: not tested

PRP (ADP) : IC₅₀>100 μM

EXAMPLE 27(9-(2-(Piperidin-4-yl)ethyl)-3,9-diazaspiro[5.5]undec-3-yl)acetic AcidTristrifluoroacetate

Step A Preparation of tert.-butyl(9-(2-(piperidin-4-yl)ethyl)-3,9-diazaspiro[5.5]undec-3-yl)acetate

0.83 g (2.22 mmol) of the pyridine from Example 20, Step G weredissolved in 50 ml dry ethanol followed by addition of 0.13 ml aceticacid and 0.1 g platinum(IV) oxide. The mixture was filled into anautoclave and hydrogenated at 50° C. and 100 atm. After three days thesame amounts of acetic acid and of the catalyst were added, but evenafter eight days the conversion was not complete. The solvent wasremoved under reduced pressure, and the residue was stirred with 5%aqueous sodium carbonate solution. It was extracted withtert.-butylmethylether, and the organic layer was dried over sodiumsulfate and concentrated in vacuo. The acetate of the title compound,which was neutralized by washing a dichloromethane solution with 2Naqueous sodium hydroxide, was obtained by chromatography on aluminumoxide with dichloromethane followed by dichloromethane/ethanol 7:3.

yield: 0.42 g (50%) beige resin

Step B: Preparation of(9-(2-(piperidin-4-yl)ethyl)-3,9-diazaspiro[5.5]undec-3-yl)acetic acidtristrifluoroacetate

0.1 g (0.263 mmol) of the ester from Step A were cleaved withtrifluoroacetic acid as described for Example 26. Step D.

yield: 0.14 g (80%) beige amorphous solid

ELISA: not tested

PRP (ADP): IC₅₀=100 μM

EXAMPLE 28(9-(2-(Piperidin-4-yl)ethyl)-2,4-dioxo-3,9-diazaspiro[5.5]undec-3-yl)aceticAcid Diacetate

To a solution of 0.5 g (1.25 mmol) of the compound from Example 26. StepC in 40 ml 90% acetic acid were added 100 mg PtO₂. The mixture wasfilled into an autoclave and hydrogenated at 20 atm and room temperaturefor 22 h. The solvent was removed under reduced pressure, and theresidue was stirred with ether. The insoluble title compound wasseparated and dried in vacuo.

yield: 0.56 g (95%) pale yellow resin

ELISA: not tested

PRP (ADP): not tested

EXAMPLE 29(S)-5-(9-(3-((Aminoiminomethyl)amino)-1-oxopropyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-benzenesulfonylamino-5-oxopentanoicAcid Hydrochloride

Step A Preparation of (S)-2-aminopentanedioic acid 1-tert.-butyl ester

To a solution of 25.0 g (74.1 mmol)(S)-2-(benzyloxycarbonylamino)pentanedioic acid 1-tert.-butyl ester(prepared according to Liebigs Ann. Chem. 1961, 646, 127) in 500 ml drymethanol were added 2.5 g 10% Pd/C, and the mixture was stirred overnight at room temperature under an atmosphere of hydrogen. The catalystwas removed by filtration through celite and the filtrate wasconcentrated under reduced pressure. The remaining solid title compoundwas purified by stirring with a small amount of ethanol, filtered withsuction, and dried in vacuo. Another crop (1.7 g) was obtained afterconcentration of the mother liquid and stirring of the residue withethyl acetate.

total yield: 11.55 g (77%), white powder, m.p. 114-116° C.

Step B Preparation of (S)-2-(benzenesulfonylamino)pentanedioic acid1-tert.-butyl ester

11.5 g (56.6 mmol) of the amino acid from the previous step weredissolved in a mixture of 710 ml water and 57 ml 1N aqueous sodiumhydroxide solution, and the solution was cooled to 0° C. 6.6 g (62.3mmol) sodium carbonate were added followed by dropwise addition of 11.0g (62.3 mmol) benzenesulfonyl chloride. After 1 h at 0° C. stirring wascontinued over night at room temperature, while the pH of the mixturedropped from pH 10 to pH 7. It was extracted two times with ethylacetate, and the aqueous layer was adjusted to pH 2 with 2N hydrochloricacid. The solution was extracted four times with ethyl acetate, and thecombined organic layers were washed with brine and with water,successively, dried over sodium sulfate, and concentrated under reducedpressure to leave the pure title sulfonamide.

yield: 13.0 g (67%) pale yellow sirup, which solidified immediately uponstanding

Step C Preparation of (S)-tert.-butyl2-benzenesulfonylamino-5-oxo-5-(9-phenylmethyl-3,9-diazaspiro[5.5]undecan-3-yl)pentanoate

A solution of 6.82 g (19.86 mmol) of the intermediate from Step B in 20ml dry THF containing 2.8 ml triethylamine was cooled to −10° C. 2.15 g(19.8 mmol) ethyl chloroformate were added dropwise, and the mixture wasstirred for ten minutes, while a precipitate was formed spontaneously. Asolution of 4.85 g (19.86 mmol) of the intermediate from Example 20,Step D in a mixture of 24 ml dry THF and 5.3 ml triethylamine was addedquickly in small portions. It was warmed to room temperature, stirredover night, poured into water, and extracted three times with ethylacetate. The combined organic layers were washed with water, dried oversodium sulfate, and concentrated under reduced pressure, and the titlecompound was obtained by chromatography on silica gel withdichloromethane/ethanol 93:7.

yield: 3.9 g (34%) colorless crystalline solid, m.p. 134-136° C.

Step D Preparation of (S)-tert.-butyl2-benzenesulfonylamino-5-(3,9-diazaspiro[5.5]undecan-3-yl)-5-oxopentanoateacetate

To 200 mg 10% Pd/C were added 30 ml dry methanol and 6 drops aceticacid. A stream of hydrogen was passed through the mixture for 20minutes, and 2.0 g (3.5 mmol) of the intermediate from the previous stepwere added. It was stirred at room temperature for four days followed byaddition of another 100 mg of the catalyst and 6 drops acetic acid, andstirring was continued for an additional day. The catalyst was removedby filtration through celite, and the filtrate was concentrated underreduced pressure to leave the title compound, which was purified bycrystallization from ethyl acetate.

yield: 1.15 g (61%) white powder, m.p. 153-156° C.

Step E Preparation of (S)-tert.-butyl2-benzenesulfonylamino-5-(9-(3-benzyloxycarbonylamino-1-oxopropyl)-3,9-diazaspiro[5.5]undecan-3-yl)-5-oxopentanoate

The reaction was carried out as described in Step C. A solution of 0.51g (2.3 mmol) 3-(benzyloxycarbonylamino)propionic acid in 3 ml THF and0.65 ml triethylamine was treated with 0.25 g (2.3 mmol) ethylchloroformate followed by a solution of 1.1 g (2.04 mmol) of thecompound from the previous step in a mixture of 4 ml THF, 0.6 mltriethylamine, and 1 ml water. It was stirred for two days at roomtemperature, and the pure title intermediate was obtained afterchromatography on silica gel with dichloromethane containing 4% ethanol.

yield: 0.86 g (62%) colorless crystals, m.p. 50-54° C.

Step F Preparation of (S)-tert.-butyl 5-(9-(3-amino-1-oxopropyl) -3,9-diazaspiro[5.5]undecan-3-yl) -2-benzenesulfonylamino-5-oxopentanoate

To a solution of 0.74 g (1.08 mmol) of the compound from Step E in 10 mlethanol were added 70 mg 10% Pd/C. It was stirred over night at roomtemperature, while a slight stream of hydrogen passed through themixture. The catalyst was removed by filtration through celite, and thefiltrate was concentrated in vacuo to leave the pure title amine.

yield: 0.56 g (94%) pale yellow foam

Step G Preparation of (S)-tert.- butyl2-benzenesulfonylamino-5-(9-(3-(((tert.-butoxycarbonylamino)(tert.-butoxycarbonylimino)methyl)amino)-1-oxopropyl)-3,9-diazaspiro[5.5]undecan-3-yl)-5-oxopentanoate

To a suspension of 0.44 g (2.03 mmol) mercury(II) oxide in 25 ml drydioxane were added successively 0.56 g (1.02 mmol) of the amine from theprevious step and 0.28 g (1.05 mmol)N,N′-bis(tert.-butoxycarbonyl)thiourea (prepared according toTetrahedron Lett. 1992, 33, 5933), each dissolved in a small amount ofdioxane. After two days stirring at room temperature another 50 mgmercury(II) oxide were added and stirring was continued for the sametime. It was filtered through a pad of celite, and the filtrate wasconcentrated under reduced pressure. The title compound was obtainedfrom the residue by chromatography on silica gel with dichloromethanecontaining 4% ethanol.

yield: 0.52 g (64%) pale yellow film, which solidified upon standing

Step H Preparation of(S)-5-(9-(3-((aminoiminomethyl)amino)-1-oxopropyl)-3,9-diazaspiro[5.5]undecan-3-yl)-2-benzenesulfonylamino-5-oxopentanoicacid hydrochloride

A stream of gaseous hydrogen chloride was passed for 40 minutes througha solution of 0.3 g (0.38 mmol) of the protected guanidine from theprevious step in 15 ml dry dichloromethane at 0° C., while a precipitatewas formed. It was stirred for additional 2 h at room temperature, andthe solvent was removed under reduced pressure. The solid residue of thetitle compound was stirrred with ether, filtered with suction, washedwith ether, and dried in vacuo at 50° C.

yield: 206 mg (95%) colorless crystalline solid, m.p.>127° C. (dec.)

ELISA: not tested

PRP (ADP): IC₅₀=

EXAMPLE 30 Preparation of(8-(4-(Aminoiminomethyl)benzoyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)aceticAcid

Step A: Preparation of t-Butyl8-Benzyl-(2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetate

A solution of 8-benzyl-2-oxo-1-oxa-3,8-diazo-spiro[4.5]decane (0.83 g,3.89 mmol), prepared as described in J. Med. Chem, 24, 1320-1328(1981),and tetrahydrofuran (5 mL) was treated with sodium hydride (0.102 g of a60% dispersion in oil, 4.28 mmol). The resulting mixture was heated toreflux. After one hour, the mixture was allowed to cool to roomtemperature. This solution was treated with t-butyl bromoacetate (0.7mL, 4.28 mmol) by dropwise addition. After one hour, the resultingmixture was diluted with ethyl acetate (50 mL) and washed with water.

The organic phase was concentrated in vacuo. The residue waschromatographed on silica gel, eluting with hexanes/ethyl acetate (1:1),to give the title compound as a solid.

Yield=1.12 g (83%), m.p. 157-159° C., FDMS m/z=360.

Step B: Preparation of t-Butyl(2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetate

A mixture of the compound prepared in Example 30A (0.60 g, 1.66 mmol),10% palladium in carbon (0.5 g), ethylacetate (200 mL), and ethanol (20mL) was stirred under a H₂ atmosphere. After four hours, the reactionmixture was filtered, and the filtrate was concentrated in vacuo.

Yield=0.41 g, m.p. 76-85° C., FDMS m/z=270.

Step C: Preparation of t-Butyl(8-(4-cyanobenzoyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetate

A solution of the compound prepared in Example 30B (0.15 g, 0.556 mmol),methylene chloride (2.0 mL), and pyridine (2.0 mL) was treated with4-cyanobenzoyl chloride (0.103 g, 0.611 mmol). After two hours at roomtemperature, the mixture was diluted with ethyl acetate (50 mL). Theresulting mixture was washed with water, and concentrated in vacuo. Theresidue was chromatographed on silica gel, eluting with ethyl acetate.

Yield=0.18 g FDMS m/z=400.3

Step D: Preparation of t-Butyl(8-(4-(N-t-Butoxycarbonyl-aminoiminomethyl)benzoyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetate

A mixture of the compound prepared in Example 30C (0.179 g, 0.45 mmol),pyridine (10 mL), and triethylamine (1 mL) was saturated with H₂S, thenallowed to stand at room temperature. After 18 hours, this mixture wasconcentrated to dryness. The residue was suspended in a mixture ofacteone (5.0 mL) and methyl iodide (5.0 mL). The resulting mixture washeated at ref lux for 1 hour. The reaction solution was then allowed tocool to room temperature and concentrated to dryness. The residue wasdisolved in methanol (5 mL), and treated with ammonium acetate (0.10 g,1.35 mmol). The resulting solution was heated at 60° C. for 2 hours.This material was concentrated to dryness. The residue was suspended ina mixture of tetrahydrofuran and water (10 mL, 1:1) and treated withpotassium carbonate (0.37 g, 2.7 mmol) and di-tert-butyl dicarbonate(0.49 g, 2.2 mmol). The resulting mixture was stirred at roomtemperature for 2 hours, then diluted with ethyl acetate (100 mL). Theresulting mixture was washed with water, then the organic phase wasconcentrated. The residue was purified by chromatography, eluting withtetrahydrofuran/chloroform (1:8) giving the title compound.

Yield=0.175 g FDMS m/z=517.

Step E: Preparation of(8-(4-(Aminoiminomethyl)benzoyl)-2-oxo-2-oxa-3,8-diaza-spiro[4.5]dec-3-yl)aceticAcid

A mixture of the compound prepared as described in Example 30D (0.175 g,0.33 mmol) and trifluoroacetic acid (10 mL) was maintained at roomtemperature for 1 hour, then concentrated to dryness. The residue wastriturated with diethyl ether, and the solid collected by filtration.

Yield=0.17 g FAB MS m/z=361.

ELISA: IC₅₀=29 μM

PRP (ADP): IC₅₀=80 μM

EXAMPLE 31 Preparation of(8-(2-(4-Aminoiminomethyl)acetyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)aceticAcid

Step A: Preparation of t-Butyl(8-(2-(4-Cyanophenyl)-acetyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetate

A mixture of the compound prepared as described in Example 30B (0.08 g,0.29 mmol), 4-cyanophenylacetic acid (0.051 g, 0.322 mmol) and methylenechloride (2.0 mL) was treated with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (0.087 g, 0.44 mmol), anddimethylaminopyridine (catalitic amount). Resulting mixture was stirredat room temperature for about 2 hours. The reaction mixture was dilutedwith ethyl acetate and washed with water. The organic phase wasconcentrated and the residue purified by chromatography, eluting withethyl acetate.

Yeild=0.11 g (85%) FABMS m/z=414.

Step B: Preparation of t-Butyl(8-(2-(4-N-t-Butoxycarbonyl-aminoiminomethyl)acetyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetate

The compound was prepared using the procedure substantially as describedin Example 30D. FDMS m/z=414.

Step C: Preparation of (8- (2- (4-Aminoiminomethyl)acetyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetic Acid

Compound was deprotected was trifluoroacetic acid following theprocedure substantially as described in Example 30E.

FABMS m/z=488.

ELISA: IC₅₀=0.074 μM

PRP (ADP) : IC₅₀=1.11 μM

EXAMPLE 32 Preparation of((9-(2-(4-(Aminoiminomethyl)phenyl)acetyl)-2-oxo-1-oxa-3,9-diaza-spiro[5.5]dec-3-yl)aceticAcid

Step A: Preparation of t-Butyl (9-Benzyl-2-oxo-1-oxa-3,9-diaza-spiro[5.5]undec-3-yl) acetate

The title compound was prepared from spirocarbonate prepared asdescribed in Eur. J. Med. Chem. Ther., 9, 416-423 (1974), using theprocedure substantially as described in Example 30A.

m.m. 113-144° C. FDMS m/z=374.

Step B: Preparation of t-Butyl(2-oxo-1-oxa-3,9-diaza-spiro[5.5]undec-3-yl)acetate

A mixture of the compound prepared as described in Step A (0.53 g), 10%palladium on carbon (0.5 g) and ethenol (10 mL) were maintained underhydrogen for 2 hours. The reaction mixture was filtered, and thefiltrated concentrate in vacuo. Recrystallization of the residue fromhexane provided the title compound

m.p. 110-115° C. FDMS m/z−284.

Step C: Preparation of t-Butyl((9-(2-(4-cyanophenyl)-acetyl)-2-oxo-1-oxa-3,9-diaza-spiro[5.5]undec-3-yl)acetate

The title compound was prepared using the procedure substantially asdescribed in Example 31A. FDMS m/z=427.

Step D: Preparation of t-Butyl((9-(2-(4-(N-t-Butoxy-carbonylaminoiminomethyl)phenyl)acetyl)-2-oxo-1-oxa-3,9-diaza-spiro[5.5]undec-3-yl)acetate

The title compound was prepared using the procedure substantially asdescribed in Example 30D. FDMS m/z=545.

Step E: Preparation of((9-(2-(4-(Aminoiminomethyl)phenyl)-acetyl)-2-oxo-1-oxa-3,9-diaza-spiro[5.5]undec-3-yl)aceticAcid

The title compound was prepared using the procedure substantially asdescribed in Example 30E.

FABMS m/z=389.

ELISA: IC₅₀=0.20 μM

(PRP): IC₅₀=2.13 μM

EXAMPLE 33 Preparation of(9-(4-(Aminoiminomethyl)benzoyl)-2-oxo-1-oxa-3,9-diaza-spiro[5.5]undec-3-yl)aceticAcid

Step A: Preparation of t-Butyl(9-(4-cyanobenzoyl)-2-oxo-1-oxa-3,9-diaza-spiro[5.5]undec-3-yl)acetate

The title compound was prepared using the procedure substantially asdescribed in Example 30C. FDMS m/z=413.

Step B: Preparation of t-Butyl(9-(4-(N-t-Butoxy-carbonylaminoiminomethyl)benxoyl)-2-oxo-1-oxa-3,9-diaza-spiro[5.5]undec-3-yl)acetate

The title compound was prepared using the procedure substantially asdescribed in Example 30D

FDMS m/z=531.

Step C: Preparation of(9-(4-(Aminoiminomethyl)benzoyl)-2-oxo-1-oxa-3,9-diaza-spiro[5.5]undec-3-yl)aceticAcid

The title compound was prepared using the procedure substantially asdescribed in Example 30E.

FABMS m/z=375.

ELISA: IC₅₀=16.5 μM

PRP (ADP): IC₅₀=66 μM

EXAMPLE 34 Preparation of(8-(4-(Aminoiminomethyl)benzyloxy)-2-oxo-1-oxa-3-aza-spiro[4.5]dec-3-yl)aceticAcid

Step A: Preparation of 8-(ethyleneketyl)-2-oxo-1-oxa-3-aza-spiro[4.5]decane-3-yl)

A solution of 1,4-cyclohexanedione mono-ethylene ketal (10 g, 64 mmol)in anhydrous methelane chloride (65 mL) was treated with trimethylsylylcyanide (9.4 mL, 70.4 mmol), and 18-crown-6 (catalitic). The mixture wasstirred at 0° C. under nitrogen for 35 minutes, then diluted with 5volumes of ethyl acetate. The organic phase was washed with saturatedsodium bicarbonate solution, then the organic phase was concentrated invacuo. The residue was dissolved in tetrahydrofuran, and the resultingsolution treated with lithium aluminun hydride. After 1 hour, thereaction mixture was worked-up using the Steinhardt procedure (Fieser &Fieser, Reagents for Organic Synthesis, volume 1, page 584). Thefiltrate was concentrated in vacuo and the resulting oil dissolved inmethanol (50 mL). This solution was treated with potassium carbonate(8.85 g, 64 mmol). After 1 hour at room temperature, the potassiumcarbonate was removed by filtration, and the filtrate concentrated invacuo. The residue was treated with diethyl carbonate (65 mL) and acatalytic amount of sodium hydride. The resulting solution was heated to125° C. and methanol collected by distillation. After all the methanolhad been distilled, a few milliliters of diethyl carbonate werecollected by distillation, then the solution was allowed to cool to roomtemperature. After dilution with ethyl acetate (10 volumes), the organicphase was washed with water and concentrated. The title compound wasrecrystallized from ethyl acetate/hexane.

Yield=5 g (37%) m.p. 182-184° C.

Step B: Preparation of t-Butyl 8-(ethyleneketal)-2-oxo-1-oxa-3-aza-spiro[4.5]dec-3-yl)acetate

The compound prepared in Step A (2.0 g, 9.38 mmol) in tetrahydrafuran(25 mL) was treated with sodium hydride (60% dispersion in oil, 0.39 g,10.3 mmol). After heating at reflux for 1 hour,t-Butyl-alpha-bromoacetate (1.32 mL, 10.3 mmol) was added to thereaction mixture. After an additional 1 hour at reflux, the solution wasallowed to cool to room temperature. This solution was diluted withethyl acetate (10 volumes), washed with water, and concentrated invacuo. The title compound was recrystallized from ethyl acetate/hexane.

Yield =2.8 g (91%) m.p. 144-146° C.

Step C: Preparation of t-Butyl8-oxo-2-oxo-1-oxa-3-aza-spiro[4.5]dec-3-yl)acetate

The compound from Step B (3.1 g, 9.47 mmol) in acetone/water (3:1) wastreated with pyridinium tosylate. After heating at 65° C. for 16 hours,the reaction was diluted with ethyl acetate (10 volumes). The organicphase was washed with 10% sodium bicarbonate, then concentrated invacuo. The title compound was recrystallized from ethyl acetate/hexane.

Yield =2.1 g (78%) m.p. 126-129° C. FDMS m/z=284.

Step D: Preparation of(8-(4-cyanobenzyloxy)-2-oxo-1-oxa-3-aza-spiro[4.5]dec-3-yl) acetate

The compound from Step C (0.25 g, 0.882 mmol) in methanol was treatedwith sodium borohydride (0.033 g, 0.882 mmol). After 2 hours undernitrogen atmosphere, the reaction mixture was concentrated in vacuo. Theresidue was diluted with tetrahydrafuran (2.5 mL), then treated withsodium hydride (60% dispersion in oil, 0.034 g, 0.882 mmol). After 1hour at room temperature under nitrogen, this mixture was treated withbromo-p-tolunitrile (0.173 g, 0.882 mmol). After an additional 72 hours,the reaction mixture was diluted with ethyl acetate. The organic phasewas washed with water and concentrated in vacuo. The residue waspurified by silica-gel chromatography, eluting with chloroform/methanol(9:1).

Yield =12 mg (3.3%)

Step E: Preparation of(8-(4-(Aminoiminomethyl)benzyloxy)-2-oxo-1-oxa-3-aza-spiro[4.5]dec-3-yl)aceticAcid

The compound from Step D (0.012 g, 0.03 mmol) was converted to the titlecompound using the procedure substantially as described in Examples 30Dand 30E.

Yield=6 mg (43%) FDMS m/z=362.

ELISA: IC₅₀=0.35 μM

PRP (ADP): IC₅₀=1.29 μM

EXAMPLE 35 Preparation of(8-(2-(4-(Aminoiminomethyl)phenyl)acetamido)-2-oxo-1-oxa-3-aza-spiro[4.5]dec-3-yl)aceticAcid

Step A: Preparation of t-Butyl(8-(2-(4-cyanophenyl)-acetamido)-2-oxo-1-oxa-3-aza-spiro[4.5]dec-3-yl)acetate

The ketone prepared as described in Example 34C was converted to thetitle compound using the procedure substantially as described in Example36A.

Yield=56 mg (15%) FDMS m/z=428.

Step B: Preparation of(8-(2-(4-(Aminoiminomethyl)phenyl)-acetamido)-2-oxo-1-oxa-3-aza-spiro[4.5]dec-3-yl)aceticAcid

The compound of Step A was converted to the title compound using theprocedure substantially as described in Example 36B.

Yield=10 mg (15%) m.p. 193-196° C. FABMS m/z=389.

ELISA: IC₅₀=0.19 μM

PRP (ADP): IC₅₀=0.77 μM

EXAMPLE 36 Preparation of(8-(4-(Aminoiminomethyl)benzamido)-2-oxo-1-oxa-3-aza-spiro[4.5]dec-3-yl)aceticAcid

Step A: Preparation of t-Butyl(8-(4-cyanobenzamido)-2-oxo-1-oxa-3-aza-spiro[4.5]dec-3-yl)acetate

The ketone prepared as described in Example 34C (0.25 g, 0.882 mmol),ammonium acetate (0.68 g, 8.82 mmol), methanol (1.5 mL), was treatedwith sodium cyanoborohydride (0.055 g, 0.882 mol). The resulting mixturewas stirred at room temperature for 16 hours, then concentrated invacuo. The residue was dissolved in water, the pH adjusted to pH 10, andthe product extracted with ethyl acetate. The combined organic extractswere concentrated in vacuo, and the residue dissolved in methylenechloride/pyridine (2.5/1.5 mL). This solution was treated with asolution of 4-cyanobenzoyl chloride (0.160 g, 0.97 mmol) in methylenechloride (1 mL). The resulting solution was stirred under nitrogen forabout 1 hour, then diluted with ethyl acetane (10 volumes). The organicphase was washed with water and concentrated in vacuo. The titlecompound was purified by silica-gel chromatography, eluting with 1%methanol in chloroform. The product was recrystallized from ethylacetate/hexane.

Yield=105 mg (30%) m.p. 200-205° C. FDMS m/z=414.

Step B: Preparation of(8-(4-(Aminoiminomethyl)benzamido)-2-oxo-1-oxa-3-aza-spiro[4.5]dec-3-yl)aceticAcid

The product from Step A was converted to the title compound using theprocedure substantially as described in Example 34E.

Yield=30 mg (65%) m.p. 280-284° C. FDMS m/z=375.

ELISA: IC₅₀=0.17 μM

PRP (ADP): IC₅₀=0.51 μM

EXAMPLE 37 Preparation of((8-(4-N-benzyloxycarbonylamino)-1-oxobutyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetate

Step A: Preparation of((8-(4-N-benzyloxycarbonylamino)l-oxobutyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetate

The amine prepared as described in Example 30B was reacted with4-(N-benzyloxycarbonylamino)butyric-acid using the procedure asdescribed in Example 38A.

Yield=169 mg (93%) FDMS m/z=490.

Step B: Preparation of t-Butyl(8-(4-(di-t-butoxycarbonyl-aminoiminomethyl)-1-oxobutyl)-2-oxo-1-oxa-3,8-diaza-spiro-[4.5]dec-3-yl)acetate

The title compound was prepared from the compound of Step A using theprocedures substantially as described in Example 38B.

Yield=160 mg (83%) FDMS m/z=598.

Step C: Preparation of((8-(4-N-benzyloxycarbonylamino)-1-oxobutyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetate

The title compound was prepared from the compound of Step B using theprocedure substantially as described in Example 38C.

Yield=90 mg (79%) FDMS m/z=342.

ELISA: IC₅₀=19 μM

PRP (ADP): IC₅₀=52 μM

EXAMPLE 38 Preparation of(8-(4-(Aminoiminomethyl)-1-oxopentyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)aceticAcid

Step A: Preparation of t-Butyl(8-(5-(N-benzyloxycarbonyl-amino)-1-oxopentyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetate

A solution of the amine prepared as described in Example 30B (0.100 g,0.37 mmol), 5-(N-benzyloxycarbonylamino)-pentolic acid (0.103 g, 0.41mmol), EDCI (0.105 g, 0.55 mmol), HOBT (0.074 g, 0.55 mmol) 4-DMAP(catalytic) and methylene chloride (2 mL) was stirred at roomtemperature for 1 hour. This solution was then diluted with ethylacetate (15 volumes). The resulting solution was washed with water andconcentrated in vacuo. The residue was purified by silica-gelchromatography, eluting with chloroform/methanol (95:5).

Yield=160 mg (86%) FDMS m/z=504.

Step B: Preparation of t-Butyl(8-(5-(di-t-Butoxycarbonyl-aminoiminomethyl)-1-oxopentyl)-2-oxo-1-oxa-3,8-diaza-spiro-[4.5]dec-3-yl)acetate

The compound from Step A (0.15 g, 0.8 mmol) and 5% palladium on carbonin absolute ethanol (10 mL) was rapidly stirred under a hydrogenatmosphere for 1 hour. The catalyst was removed by filtration and thefiltrate concentrated in vacuo. The residue was treated withbis-Boc-thiourea (0.082 g, 0.298 mmol), triethylamine (0.14 mL, 0.983mmol) mercury (II) chloride (0.89 g, 0.328 mmol), and dimethylformamide(2mL). The resulting mixture was stirred at 0° C. for 1½ hours, then atroom temperature for 1 hour. This mixture was diluted with ethyl acetate(15 volumes). The resulting mixture was washed with water andconcentracted in vacuo. The residue was purified by silica-gelchromatography, eluting with chloroform/methanol (97:7).

Yield=160 mg (88%) FDMS m/z=612.

Step C: Preparation of(8-(4-(Aminoiminomethyl)-1-oxopentyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)aceticAcid

The compound from Step B (0.15 g, 0.245 mmol) was treated withtrifluoroacetic acid (5 mL). After 1 hour at room temperature, thesolution was concentrated in vacuo. The residue was dissolved in waterand lyophilized.

Yield=100 mg (88%) FABMS m/z=356.

ELISA: IC₅₀=1.85 μM

PRP (ADP): IC₅₀=7 μM

EXAMPLE 39 Preparation of(8-(6-(aminoiminomethyl)-1-oxohexyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)aceticAcid

Step A: Preparation of t-Butyl(8-(6-(benzyloxycarbonyl-amino)-1-oxohexyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetate

The title compound was prepared from the amine of(6-(N-benzyloxycarbonylamino)hexoic acid substantially as described inExample 38A.

Yield=230 mg (73%) FDMS m/z=518.

Step B: Preparation of t-Butyl(8-(6-(di-t-Butoxytoxy-carbonylaminoiminomethyl)-1-oxohexyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)acetate

The compound was prepared from the compound of Step A using theprocedure substantially as described in Example 38B.

Yield=250 mg (98%) FDMS m/z=626.

Step C: Preparation of(8-(6-(aminoiminomethyl)-1-oxohexyl)-2-oxo-1-oxa-3,8-diaza-spiro[4.5]dec-3-yl)aceticAcid

The compound was prepared from the compound of Step B using theprocedure substantially as described in Example 38C.

Yield=156 mg (85%), FABMS m/z=370.

ELISA: IC₅₀=2.7 μM

PRP (ADP): IC₅₀=12 μM

EXAMPLE 40 Preparation of2-(tert-butoxycarbonyl)-2,9-diaza-spiro[5.5]undecane:

The synthesis of the 2,9-diaza-spiro[5.5]undecane template wasaccomplished by slightly modifying the procedure utilized for thepreparation of the 3,9-diaza-spiro[5.5]undecane as described in U.S.Pat. No. 5,451,578.

Step A

Commercially available 1-benzyl-3-piperidone hydrochloride hydrate (1.89g) was dissolved in H₂O (10 mL) and then 1M NaOH (9 mL) was added. Thesolution was stirred at rt for 5 minutes. Extraction with CH₂Cl₂ (3×20mL), drying with Na₂SO₄, and removal of solvent under vacuum afforded1.52 g of the neutralized material.

This material was then dissolved in ice cold EtOH (15 mL) which wassaturated with NH₃. Then ethyl cyanoacetate (1.7 mL) was added viasyringe. The resulting solution was let standing at 0° C. overnight.Evaporation of the solvent gave 2.8 g of a viscous syrup. This syrup wasdissolved in H₂O (11 mL) containing concentrated H₂SO₄ (13 mL) and theresulting solution was gently refluxed for 3 days. Removal of most ofthe H₂O under vacuum was followed by azeotropic removal of the rest ofthe H₂O using EtOH (4×50 mL). The product was then dissolved in EtOH (50mL) and refluxed overnight. Most of the EtOH was then evaporated. Aftercooling to 0° C., H₂O (25 mL) added, and then K₂CO₃ (19 g) was carefullyadded with vigorous stirring. The mixture was carefully diluted with H₂O(200 mL) and washed with CH₂Cl₂ (2×150 mL). After the organics weredried with Na₂SO₄, the solvent was evaporated to afford 2.01 g (69%yield) of the desired diester which was used without furtherpurification.

MS(ES): (M+H)⁺=348

Step B

The diester (1.93 9) was dissolved in THF (10 mL) and cooled to −40° C.A commercially available solution of LAH in Et₂O (1M, 8.9 mL) was thenslowly added via syringe followed by stirring for 40 minutes as itwarmed to rt. After the reaction was cooled to 0° C., sequentialaddition of H₂O (0.35 mL), 1M NaOH (0.7 mL) and H₂O (0.8 mL) followed.The suspension was stirred vigorously for 1 h at 0° C., diluted withEt₂O (20 mL), and filtered. After thorough rinsing of the solid withEt₂O (150 mL), the solvent was removed under vacuum to afford 1.38 g ofthe desired diol (94% yield) which was used without furtherpurification.

MS(ES): (M+H)⁺=264

Step C

A solution of the N-benzyl diol (1.3 g), di-tert-butyl dicarbonate (1.1g), and 10% Pd(OH)₂/C (130 mg) in MeOH (35 mL) was stirred at 50 psi ofH₂ overnight. The mixture was filtered through celite and rinsedthoroughly with MeOH. Evaporation of the solvent afforded 859 mg (64%yield) of the desired carbamate which was used without furtherpurification.

MS(ES): (M+H)⁺=274

Step D

The carbamate (825 mg) was dissolved in anhydrous CH₂Cl₂ (15 mL) underan argon atmosphere and cooled to −20° C. To this solution was addedEt₂N (1.23 mL) and then methanesulfonyl chloride (0.51 mL). The solutionwas stirred for 1 h, then poured into ice cold 1 M citric acid (40 mL).This mixture was washed with Et₂O (4×30 mL). The combined organic washeswere then 10 extracted with saturated NaHCO₃ (1×30 mL), dried with MgSO₄and evaporated to give 604 mg (47% yield) of the di-mesylateintermediate which was used without further purification.

MS(ES): (M+H)⁺=430

The di-mesylate (600 mg) was dissolved in MeOH (10 mL). ConcentratedNH₄OH (5 mL) was added and the rection vessel was sealed stirred at 55°C. overnight. After cooling to 0° C., the solution was poured into icecold 0.5M NaOH (40 mL). Washing with EtOAc (3×50 mL), drying theorganics with K₂CO₃ and solvent evaporation afforded 210 mg of thedesired spirocycle.

MS(ES): (M+H)⁺=254

EXAMPLE 41 Preparation of4-((3-(4-aminoiminomethyl)benzoyl)-3,9-diaza-spiro[5.5]undec-9-yl)-4-oxobutanoicacid

Step A: Preparation of3-tert-butoxycarbonyl-9-(4-cyanobenzoyl)-3,9-diaza-spiro[5.5]undecane

The synthesis of the starting material,3-tert-butoxycarbonyl-3,9-diaza-spiro[5.5]undecane, was accomplished asdescribed in U.S. Pat. No.5,451,578. This material (1.45 g) wasdissolved in CH₂Cl₂ (25 mL) and cooled to 0° C. under an argonatmosphere. To this solution was added triethylamine (1.67 mL) and4-dimethylaminopyridine (70 mg) followed by 4-cyanobenzoyl chloride(1.04 g). The mixture was stirred overnight as it warmed to rt. It wascooled to 0° C., and ice cold 1M HCl (30 mL) was carefully added. Theresulting mixture was washed with EtOAc (3×30 mL). Then washing theEtOAc with saturated NaHCO₃ and brine followed by drying with MgSO₄ andevaporation of the solvent under reduced pressure afforded the titlecompound (1.62 9, 74%) as a yellow foam. Chromatography on silica gelwith hexanes/ethyl acetate (2:1) as eluant gave pure compound.

MS(ES): (M+H)⁺=384.

Step B: Preparation of4-(3-(4-cyanobenzoyl)-3,9-diaza-spiro[5.5]undec-9-yl)-4-oxobutanoic acid

To the material obtained in step A (100 mg) was added 40% TFA in CH₂Cl₂(2.5 mL) at rt. After stirring for ½ h, the solvent was evaporated. Theresultant residue was dissolved in anhydrous CH₂Cl₂ (3 mL) under argonand cooled to 0° C. After adding triethylamine (0.13 mL), succinicanhydride (30 mg) in CH₂Cl₂ (1.4 mL) was added. Stirring was continuedovernight as it warmed to rt. The solvent was then evaporated and theresidue was partitioned between 1M HCl (15 mL) and EtOAc (4×15 mL). Thecombined organics were washed with brine (30 mL), dried with MgSO₄, andevaporated to afford 73 mg (74% yield) of the desired acid.

MS(ES): (M+H)⁺=384.

Step C: Preparation of the title compound

H₂S gas was bubbled gently for 10 minutes into a pyridine (4 mL)solution of the cyano acid (70 mg) obtained in part B containingtriethylamine (0.22 mL). After the resultant solution was stirred at rtovernight, argon was bubbled through the solution for ½ h to removeexcess H₂S. This pyridine solution was poured into 1M HCl (50 mL) andthen saturated with NaCl. This solution was then washed with EtOAc (6×20mL). The combined EtOAc was dried over MgSO₄ and evaporated to give 73mg of a yellow semi-solid.

The yellow semi-solid was suspended in acetone (10 mL) and then methyliodide (3 mL) was added. After stirring overnight at rt the solvent wasevaporated to give 103 mg of a yellow semi-solid.

This material was then dissolved in EtOH (15 mL) and then ammoniumacetate (2 g) was added. The resultant solution was stirred at 60° C.overnight. Evaporation of the solvent afforded crude product which wasthen purified using reversed phase high pressure liquid chromatography(RP-HPLC).

MS(ES): (M+H)⁺=401

ELISA: IC₅₀=15 μM.

PRP (ADP): IC₅₀=14 μM.

EXAMPLE 42 Preparation of5-((3-(4-aminoiminomethyl)benzoyl)-3,9-diaza-spiro[5.5]undec-9-yl)-5-oxopentanoicacid

The title compound was prepared by substantially following the procedurein Example 40 except that glutaric anhydride was used in place ofsuccinic anhydride in step B.

MS(ES): (M+H)⁺=415

ELISA: IC₅₀=3.5 μM.

PRP (ADP): IC₅₀=10 μM.

EXAMPLE43 Preparation of Ethyl(3-(4-Aminoiminomethyl)benzoyl)-2-aza-spiro[5.5]undec-9-yl)acetatehydrochloride

Step A: Preparation ofbenzyl-9-oxo-2-aza-spiro[5.5]undec-7-ene-3-carboxylate

A solution of benzyl-3-formylpiperidine-1-carboxylate (1 mmol) andmethylvinyl ketone (1.2 mmol) was dissolved in MeOH (1 mL) and H₂O (1mL). To this added catalytic amount of KOH and the solution was refluxedfor 1 hour. To this then added powdered molecular sieves and refluxedfor another 1 h. The HPLC analysis showed the completion of thereaction. The molecular sieves are filtered and the filtrate wasevaporated. The residue was disssolved in CH₂Cl₂ and washed with water,dried, filtered and evaporated to give the desired spirocyclic enone asa yellow oil in 60% yield.

Step B: Preparation ofbenzyl-9-(ethoxycarbonyl)methylene)2-aza-spiro[5.5]undec-7-ene-3-carboxylate

To the suspension of sodium hydride (3mmol) in THF ( 2 mL) at 0° C. wasadded triethylphoshonoacetate and then stirred for 30 min at 0° C. Asolution of spiro compound (1 mmol) from previous step in THF (2 mL),and the reaction was heated to 50° C. for Bh. The mixture was pouredinto water and extracted with ethylacetate. The title compound waspurified by flash chromatography on silica gel using ethylacetate/hexaneto give the desired pure compound as a colorless oil (50% )

Step C: Preparation of ethyl(2-aza-spiro[5.5]undec-9-yl)acetate

The protected spiro compound from Step B was dissolved in 10 mL ofethanol, and to this added Pd(II) hydroxide on carbon. The reaction wascarried at 50 psi H2 pressure for 18 h. The catalyst was removed byfiltering through celite, and the filtrate concentrated to give oil in70% yield.

Step D: Preparation ofethyl(3-(4-cyanobenzoyl)-2-aza-spiro[5.5]undec-9-yl)acetate

The 1 mmol of compound from previous step and triethylamine (3 mmol)were dissolved in dry CH₂Cl₂. To this added 4-cyanobenzoyl chloride (1.1mmol) and solution was stirred for 12 hours at room temperature. Thesolvent was evaporated and the residue treated with water and extractedthe product with ethylacetate. The organic layer was washed with 10% HClsolution, dried, and solvent removed to afford crude product. Theproduct was purified on reverse phase HPLC to afford desired material asa yellow oil (60%).

Step E: Preparation ofethyl(3-(4-(hydroxyiminomethyl)benzoyl)-2-aza-spiro[5.5]undec-9-yl)acetatehydrochloride

To a solution of nitrile (1 mmol) in ethanol (5 mL) was addedhydroxylamine hydrochloride (2mmol) and triethylamine (2 mmol). Thereaction mixture was heated to 65° C. and stirred for 3 hours. Thesolvent was evaporated and diluted with water and extracted the productwith ethylacetate. The organic layer was dried, filtered and evaporatedto afford hydroxyamidino compound as a white solid.

Step F: Preparation ofethyl(3-(4-aminoiminomethyl)benzoyl)-2-aza-spiro[5.5]undec-9-yl)acetate.

To a suspension of the product from Step E in HOAc was added 5% Pd/C(50% wet). The mixture was heated at 50° C. using 45 psi H2 for 18hours. The catalyst was filtered and the solvent evaporated underreduced pressure. The crude residue from this step was taken to nextstep without further purification

Step G: Preparation of(3-(4-aminoiminomethyl)benzoyl)-2-aza-spiro[5.5]undec-9-yl)acetic acid

50 mg of the ester from step F was treated with LiOH (4 eq) in H2O andTHF.

The solution was stirred overnight, HPLC analysis showed completion ofthe reaction. The solvent was evaporated and the residue was purified onreverse phase HPLC to afford the desired pdt as a white solid in 50%yield.

ESMS: 358(MH+)

EXAMPLE 44 Preparation of (3-(4-(Aminomethyl)benzoyl)3-aza-spiro[5.5]undec-9-yl)propionic acid

Step A Preparation of benzyl -9-(ethoxycarbonyl)ethylene)3-aza-spiro[5.5]undec-7-ene-3-carboxylate

The spiro ketone (1 mmol), synthesized fromN-carbobenzyloxy-4-formylpiperidine following the same procedure as inExample 1 (Step A) in THF was added to THF solution of triethylphosphonopropionate and sodium hydride. The reaction mixture was heatedat 50° C. for 8 h, followed by addition of water and extracting theproduct with dichloromethane. The dichloromethane layer was dried,filtered, evaporated to give light yellow oil. This was purified bysilica gel chromatography to give colorless oil as the desired product

Step B: Preparation of ethyl(3-aza-spiro[5.5]undec-9-yl)propionate

The protected spiro compound from Step A was dissolved in 10 mL ofethanol, and to this added Pd(II) hydroxide on carbon. The reaction wascarried at 50 psi H2 pressure for 18 h. The catalyst was removed byfiltering through celite, and the filtrate concentrated to give oil.

Step C: Preparation ofethyl(3-(4-cyanobenzoyl)-3-aza-spiro[5.5]undec-9-yl)propionate

The 1 mmol of compound from previous step and triethylamine (3 mmol)were dissolved in dry CH₂Cl₂. To this added 4-cyanobenzoyl chloride (1.1mmol) and solution was stirred for 12 hours at room temperature. Thesolvent was evaporated and the residue treated with water and extractedthe product with ethylacetate. The organic layer was washed with 10% HClsolution, dried, and solvent removed to afford crude product. Theproduct was purified on reverse phase HPLC as a yellow oil.

Step D: Preparation ofethyl(3-(4-(hydroxyiminomethyl)benzoyl)-3-aza-spiro[5.5]undec-9-yl)propionatehydrochloride

To a solution of nitrile (1 mmol) in ethanol (5 mL) was addedhydroxylamine hydrochloride (2 mmol) and triethylamine (2 mmol). Thereaction mixture was heated to 65° C. and stirred for 3 hours. Thesolvent was evaporated and diluted with water and extracted the productwith ethylacetate. The organic layer was dried, filtered and evaporatedto afford hydroxyamidino compound as a white solid.

Step E: Preparation ofethyl(3-(4-aminoiminomethyl)benzoyl)-3-aza-spiro[5.5]undec-9-yl)propionate.

To a suspension of the product from Step E in HOAc was added 5% Pd/C(50% wet). The mixture was heated at 50° C. using 45 psi H₂ for 18hours. The catalyst was filtered and the solvent evaporated underreduced pressure. The crude residue from this step was taken to nextstep without further purification

Step F: Preparation of(3-(4-aminoiminomethyl)benzoyl)-3-aza-spiro[5.5]undec-9-yl)propionicacid

50 mg of the ester from step F was treated with LiOH (4 eq) in H2O andTHF. The solution was stirred overnight, HPLC analysis showed completionof the reaction. The solvent was evaporated and the residue was purifiedon reverse phase HPLC to afford the desired pdt as a white solid.

EXAMPLE 45 Preparation of(3-(4-Aminoiminomethyl)benzoyl)-3-aza-spiro[5.5]undec-9-carbonyl)formicacid hydrochloride

Step A Preparation of 3-(2[(3-Azaspiro[5.5]undecane-9-diethyl ester

1-Benzyl-4-piperidone was converted to the bismesylate as described in(see U.S. Pat. No. 5,451,578 for detailed procedure). To a slurry of 60%NaH (3 mmol) in toluene (10 mL)under argon, was slowly added diethylmalonate (1.5 mmol). The mixture was cooled to 0° C. and bis-mesylate (1mmol) was added and the mixture heated to reflux for 18 h. The reactionwas quenched into 10% citric acid and product extracted withdichloromethane. The crude residue was purified by flash chromatographyto give the desired diester in 65% yield.

Step B. Preparation of 3-{2[(3-Azaspiro[5.5]undecane-9-carbonyl)formicacid

To a solution of diester (300 mg) in THF (5 mL) is added 1 N LiOH (4 mL)and stirred the reaction for 3 days at room temperature. The pH wasadjusted to 2.5 and product extracted with ethylacetate. This mixturewas redissolved in THF and added 1N LiOH and the reaction was heated at80° C. overnight. The pH was again adjusted to 2 and the desired acidextracted with ethylacetate. The organic layer was dried, filtered andevaporated to give desired Boc-acid as white foam (200 mg, 80%).

Step C: Preparation of(3-(4-cyano)benzoyl)-3-aza-spiro[5.5]undec-9-carbonyl)formic acidhydrochloride

To a solution of the BOC-acid (150 mg) in CH₂Cl₂ (2 mL) at 0° C. isadded TFA(2 mL). The solution is stirred at 0° C. for 2 h. The solventand excess TFA are evaporated to an oily residue, which was used as suchfor the acylation step. The acid (1 mmol) is dissolved in CH₂Cl₂ (5 mL),added triethylamine (2 mmol) followed by p-cyanobenzoyl chloride (1.05mmol). The solution was stirred for 12 hours at room temperature. Thesolvent was evaporated and residue was suspended in water and extractedthe product with ethyl acetate. The organic layer is dried, filtered andevaporated to give crude product. The desired acylated material wasisolated as a pure material after silica gel chromatography inapproximately 60% yield.

ES-MS: 327 (M+H+)

Step D: Preparation of(3-(4-Aminoiminomethyl)benzoyl)-3-aza-spiro[5.5]undec-9-carbonyl)formicacid hydrochloride

A solution The nitrile as dissolved in a mixture of pyridine andtriethylamine, and this was saturated with hydrogen sulfide,left at roomtemperature overnight. Then poured into water and extracted withethylacetate. The ethylacetate layer was concentrated and to this addedacetone and methyl iodide followed by reflux for 2 h. After thatconcentrated the precipitate, dissolved in ethanol, treated withammonium acetate and heated for 2 h at 60° C. The solvent was removed togive the amidine functionality.

The following assay methods are suitable for evaluating the compounds ofthe invention.

No. 1—The ELISA IIb-IIIa Assay

In the following assay, GPIIb-IIIa is prepared in purified form, by amethod such as described by Fitzgerald, L. A., et al., Anal Biochem(1985) 15:169-177, (the disclosure of which is incorporated herein byreference). GPIIb-IIIa is coated onto microtiter plates. The coatedsupport is then contacted with fibrinogen and with the test material(e.g., compounds of Formula I) and incubated for a sufficient time topermit maximal binding of fibrinogen to the immobilized GPIIb-IIIa.Fibrinogen is typically provided at a concentration of about 5-50 nM andthe test material can, if desired, be added at a series of dilution.Typical incubations are 2 to 4 hours at 25° C., the time and temperaturebeing interdependent.

After incubation, the solution containing the fibrinogen and testmaterial is removed and the level of binding of fibrinogen measured byquantitating bound fibrinogen to GPIIb-IIIa. Any suitable means ofdetection may be used, but it is convenient to employ labeledfibrinogen, for example using biotinylated labels. Such methods are wellknown in the art.

A. Description of Assays—Plate Assay

Purified platelet GPIIb-IIIa receptor was prepared as described byFitzgerald, L. A., et al., Anal Biochem (1985) 151:169-177 (1985).Vitronectin receptor was prepared as described by Smith, J. W., J. BiolChem (1988) 263:18726-18731. After purification, the receptors werestored in 0.1% Triton X-100 at 0.1-1.0 mg/ml.

The receptors were coated to the wells of 96-well flat-bottom ELISAplates (Linbro EIA-Plus microtiter plate, Flow Laboratories) afterdiluting 1:200 with a solution of 20 mM Tris-HCl, 150 mM NaCl, 1 mMCaCl₂, pH 7.4, to reduce the Triton X-100 concentration to below itscritical micellar concentration and adding an aliquot of 100 ul to eachwell. The wells were all allowed to incubate overnight at 4° C., andthen aspirated to dryness. Additional sites were blocked by the additionof bovine serum albumin (BSA) at 35 mg/ml in the above buffer for twohours at 30° C. to prevent nonspecific binding. The wells were thenwashed once with binding buffer (50 nM Tris-HCl, 100 mM NaCl 2 mM CaCl₂,1 mg/ml BSA).

The corresponding ligands (fibrinogen, von Willebrand Factor, orvitronectin) were conjugated to biotin using commercially availablereagents and standard protocols. The labeled ligands were added to thereceptor-coated wells at final concentration of 10 nM (100 ul/well) andincubated for 3 hours at 25° C. in the presence or absence of the testsamples. After incubation, the wells are aspirated to dryness and boundligand is quantitated.

The bound protein is detected by the addition of antibiotin antibodyconjugated to alkaline phosphatase followed by addition of substrate(p-nitrophenyl phosphate), and determination of the optical density ofeach well at 405 nM. Decreased color development is observed in wellsincubated with test samples which inhibit binding of ligand to receptor.

No. 2—The Platelet Aggregation Assay

In addition to the ELISA IIb-IIIa assay previously described theAggregation-Human PRP/ADP Assay is useful for evaluating therapeuticcompounds.

Platelet-rich plasma was prepared from healthy human volunteers for usein determining inhibition of platelet aggregation by the compounds.Blood was collected via a 21-gauge butterfly cannula, using atwo-syringe technique into {fraction (1/10)} volume of 3.8% trisodiumcitrate.

Platelet-rich plasma was prepared at room temperature by centrifugationof the citrated whole blood at 100×g for twelve minutes. The plateletrich plasma contained approximately 200-400,000 platelets/μl.

Platelet-poor plasma was prepared by centrifugation of citrated wholeblood at 12,000×g for 2 minutes.

Platelet aggregation was assayed in a 4-channel platelet aggregationprofiler (PAP-4, Biodata, Hatboro, Pa.) according to the manufacturersdirections. Inhibition of platelet aggregation was studied by addingvarying amounts of adenosine diphosphate (ADP) to stirred humanplatelet-rich plasma. Specifically, the human platelet-rich plasma wasincubated with the compound being tested for 1 minute at 37° C. prior tothe addition of a variety of aggregating agents most often ADP 5 μM, butalso 1 μg/ml collagen, 1 μM U46619 and 0.3 μM platelet activatingfactor.

Pharmaceutical Compositions

Pharmaceutical formulations containing compounds of the invention can beadministered orally in the form of tablets, capsules, solutions,emulsions or suspensions, inhaled liquid or solid particles, as a spray,through the skin by an appliance such a transdermal patch (such asdescribed in U.S. Pat. Nos. 5,296,222 and 5,271,940, the disclosures ofwhich are incorporated herein by reference) or rectally, for example, inthe form of suppositories. The lipophilic prodrug derivatives of theinvention are particularly well suited for transdermal absorptionadministration and delivery systems. Administration can also take placeparenterally, for example in the form of injectable solutions.

Tablets are prepared by mixing the Active Ingredient (“ActiveIngredient” is one or more spiro bicyclic compounds of the inventioninclusive of those corresponding to formulae I) with pharmaceuticallyinert, inorganic or organic carriers, diluents, and/or excipients.Examples of such excipients which can be used for tablets, are lactose,maize starch or derivatives thereof, talc, stearic acid or saltsthereof. Examples of suitable excipients for soft gelatin capsules arevegetable oils, waxes, fats, semisolid and liquid polyols.

Suitable excipients for the preparation of solutions and syrups arewater, polyols, sucrose, invert sugar and glucose.

Suitable excipients for injectable solutions are water, alcohols,polyols, glycerol and vegetable oils.

These pharmaceutical products can additionally contain preservatives,solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners,colorants, flavorings, buffers, coating agents and antioxidants.

Pharmaceutical compositions of this invention for parenteral injectioncomprise pharmaceutically-acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions as well as sterilepowders for reconstitution into sterile injectable solutions ordispersions just prior to use.

The Active Ingredient can also be made in micro-encapsulated form.

Exemplary formulations using the Active Ingredient are described below:

Formulation 1

Hard gelatin capsules are prepared using the following ingredients:

(mg/capsule) Active Ingredient 250.0 Starch 305.0 Magnesium stearate 5.0

The above ingredients are mixed and filled into hard gelatin capsules in560 mg quantities.

Formulation 2

A tablet formula is prepared using the ingredients below:

(mg/tablet) Active Ingredient 250.0 Cellulose, microcrystalline 400.0Colloidal silicon dioxide 10.0 Stearic acid 5.0

The components are blended and compressed to form tablets, each weighing665 mg.

Formulation 3

A dry powder inhaler formulation is prepared containing the followingcomponents:

Weight % Active ingredient 5 Lactose 95

The active mixture is mixed with the lactose and the mixture is added toa dry powder inhaling appliance.

Formulation 4

Tablets, each containing 60 mg of active ingredient, are prepared asfollows:

(milligrams) Active ingredient 60.0 Starch 45.0 Microcrystallinecellulose 35.0 Polyvinylpyrrolidone (as 10% solution in water) 4.0Sodium carboxymethyl starch 4.5 Magnesium stearate 0.5 Talc 1.0 Total150.0

The active ingredient, starch and cellulose are passed through a No. 20mesh U.S. sieve and mixed thoroughly. The solution ofpolyvinylpyrrolidone is mixed with the resultant powders, which are thenpassed through a 16 mesh U.S. sieve. The granules as produced are driedat 50-60° C. and passed through a 16 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate, and talc, previously passedthrough a No. 30 mesh U.S. sieve, are then added to the granules which,after mixing, are compressed on a tablet machine to yield tablets eachweighing 150 mg.

Formulation 5

Capsules, each containing 80 mg of medicament are made as follows:

(milligrams) Active ingredient 80.0 Starch 109.0 Magnesium stearate 1.0Total 190.0

The active ingredient, cellulose, starch, and magnesium stearate areblended passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 190 mg quantities.

Formulation 6

Suppositories, each containing 225 mg of active ingredient are made asfollows:

Active Ingredient 225 mg Saturated fatty acid glycerides to 2000 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation 7

Suspensions, each containing 50 mg of medicament per 5.0 mL dose aremade as follows:

Active ingredient 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethylcellulose (11%) Microcrystalline cellulose (89%) 50.0 mg Sucrose 1.75 gSodium benzoate 10.0 mg Flavor q.v. Color q.v. Purified water to 5.0 mL

The medicament, sucrose and xanthan gum are blended, passed through aNo. 10 mesh U.S. sieve, and then mixed with a previously made solutionof the microcrystalline cellulose and sodium carboxymethyl cellulose inwater. The sodium benzoate, flavor, and color are diluted with some ofthe water and added with stirring. Sufficient water is then added toproduce the required volume.

Formulation 8

Capsules, each containing 150 mg of medicament, are made as follows:

(milligrams) Active ingredient 150.0 Starch 407.0 Magnesium stearate 3.0Total 560.0

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 20 mesh U.S. sieve, and filled into hardgelatin capsules in 560 mg quantities.

Method of Treatment

This invention provides a method of preventing or treating thrombosis inmammals, especially humans, which method comprises administering to thehuman or mammal a therapeutically effective amount of the compounds ofthis invention. The platelet aggregation inhibitors of the invention areuseful therapeutically to prevent thrombus formation. Indicationsappropriate to such treatment include, without limitation,atherosclerosis and arteriosclerosis, acute myocardial infarction,chronic unstable angina, transient ischemic attacks and strokes,peripheral vascular disease, arterial thrombosis, preeclampsia,embolism, restenosis and/or thrombosis following angioplasty, carotidendarterectomy, anastomosis of vascular grafts, and chroniccardiovascular devices (e.g., in-dwelling catheters or shunts“extracorporeal circulating devices”). These syndromes represent avariety of stenotic and occlusive vascular disorders thought to beinitiated by platelet activation on vessel walls.

The PAIs may be used for prevention or abortion of arterial thrombusformation, in unstable angina and arterial emboli or thrombosis, as wellas treatment or prevention of myocardial infarction (MI) and muralthrombus formation post MI. For brain-related disorders, treatment orprevention of transient ischemic attack and treatment of thromboticstroke or stroke-in-evolution are included.

The PAIs may also be used for prevention of platelet aggregation,embolization, or consumption in extracorporeal circulations, includingimproving renal dialysis, cardiopulmonary bypasses, hemoperfusions, andplasmapheresis.

PAIs prevent platelet aggregation, embolization, or consumptionassociated with intravascular devices, and administration results inimproved utility of intraaortic balloon pumps, ventricular assistdevices, and arterial catheters.

The PAIs will also be useful in treatment or prevention of venousthrombosis as in deep venous thrombosis, IVC, renal vein or portal veinthrombosis, and pulmonary venous thrombosis.

Various disorders involving platelet consumption, such as thromboticthrombocytopenic purpura are also treatable.

In addition, the PAIs of the present invention may be used in numerousnontherapeutic applications where inhibiting platelet aggregation isdesired. For example, improved platelet and whole blood storage can beobtained by adding sufficient quantities of the compounds, the amount ofwhich will vary depending upon the length of proposed storage time, theconditions of storage, the ultimate use of the stored material, etc.

Preferably, the compounds of this invention are administered in the formof a pharmaceutical formulation. Thus, the compounds of this inventionmay be administered orally, parenterally, topically, rectally and etc.,in, appropriate dosage units, as desired.

The term parenteral as used herein includes subcutaneous, intravenous,intraarterial, injection or infusion techniques, without limitation. Theterm, “topically” encompasses administration rectally and by inhalationspray, as well as the more common routes of the skin and the mucousmembranes of the mouth and nose.

Actual dosage levels of active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to administer anamount of the active compound(s) that is effective to achieve thedesired therapeutic response for a particular patient.

The selected dosage level will depend upon the activity of theparticular compound, the route of administration, the severity of thecondition being treated, and the condition and prior medical history ofthe patient being treated. However, it is within the skill of the art tostart doses of the compound at levels lower than required to achieve thedesired therapeutic effect and to gradually increase the dosage untilthe desired effect is achieved. If desired, the effective daily dose maybe divided into multiple doses for purposes of administration, e.g., twoto four separate doses per day. It will be understood, however, that thespecific dose level for any particular patient will depend upon avariety of factors including the body weight, general health, diet, timeand route of administration, combination with other drugs and theseverity of the particular disease being treated.

The range of therapeutic dosages is from about 0.01 to about 10,000milligrams per day, with from 1 to 300 milligrams being preferred.

Many modifications and variations of this invention may be made withoutdeparting from its scope, as is apparent to those skilled in the art.The specific embodiments described herein are offered by way of exampleonly, and the invention is to be limited only by the terms of theappended claims.

What is claimed is:
 1. A compound of formula (I):

wherein; the spirocycle comprising (A_(i))_(p), C, and (B_(j))_(q) isselected from the formulae:

m is a number from zero to 6; R₁₀ is the same or different and is anon-interfering substituent independently selected from alkyl,halosubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl,hydroxy, alkoxy, arylalkoxy, amino, substituted amino, carbamoyl,carboxy, acyl, cyano, halo, nitro, or sulfo; n is a number from zero to6; R₀ is the same or different and is a non-interfering substituentindependently selected from alkyl, halosubstituted alkyl, alkenyl,alkynyl, cycloalkyl, aryl, arylalkyl, hydroxy, alkoxy, arylalkoxy,amino, substituted amino, carbamoyl, carboxy, acyl, cyano, halo, nitro,or sulfo; where Q—(L) is attached at a, and R₃ is attached at b; thelinking group —(L)— is a bond or a substituted or unsubstituted chainselected from the group consisting of CO, CO(C₁-C₆ alkyl), O(C₁-C₆alkyl), NHCO, and C₁-C₆ alkyl; R₂ is hydrogen, C₁-C₆ alkyl, aryl,substituted aryl, or arylalkyl; Q is a basic group selected from thegroup consisting of amino, imino, amidino, hydroxyamidino,N-alkylamidine, N,N′-dialkylamidine, N-arylamidine, aminomethyleneamino,iminomethylamino, guanidino, aminoguanidino, alkylamino, dialkylamino,trialkylamino, alkylideneamino, pyrrolyl, imidazolyl, pyrazolyl,pyridyl, pyrazinyl, pyrimidinyl, indolizinyl, isoindolyl, 3H-indolyl,indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl,phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl,amido, thioamido, benzamidino, pteridinyl, 4aH-carbozolyl, carbozolyl,beta-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl,phenanthrolinyl, phenazinyl, phenarsazinyl, phenothiazinyl, pyrrolinyl,imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, any ofthe foregoing radicals substituted on a benzene ring, and any of theforegoing radicals substituted by amino, imino, amidino, hydroxyamidino,aminomethyleneamino, iminomethylamino, guanidino, alkylamino,dialkylamino, trialkylamino, tetrahydroisoquinolinyl, dihydroisoindolyl,alkylideneamino or

R₃ is an acidic group selected from the group consisting of CO₂R₅,(C₁-C₆ alkyl)CO₂R₅, CO(C₁-C₆ alkyl)CO₂R₅, CONH(C₁-C₆ alkyl)CO₂R₅, (C₁-C₆alkyl)CH(NHR₄)CO₂R₅, CO(C₁-C₆ alkyl)CH(NHR₄)CO₂R₅, or CONH(C₁-C₆alkyl)CH(NHR₄)CO₂R₅, wherein R₄ is SO₂(C₁-C₆ alkyl), SO₂ aryl, orSO₂(substituted aryl); and R₅ is hydrogen, C₁-C₆ alkyl, aryl, orsubstituted aryl; or a pharrnaceutically-acceptable salt, solvate orprodrug thereof.
 2. The compound of claim 1 wherein Q is pyridin-4-yl,piperidin-4-yl, amidino, hydroxyamidino, guanidinyl, or a group of theformula:

wherein R₂₀ is hydrogen or halogen.
 3. The compound of claim 2 whereinthe spirocycle is selected from the formulae:

where m is one.
 4. The compound of claim 2 wherein the spirocycle isselected from the formulae:

where m is one, and R₂ is as defined in claim
 1. 5. The compound ofclaim 2 wherein the spirocycle comprising (A_(i))_(p), C, and(B_(j))_(q) is selected from the formulae:

where R₂ is hydrogen, C₁-C₆ alky, aryl, substituted aryl, or arylalkyl.6. The compound of claim 1 wherein R₃ is CO₂R₅, (C₁-C₆ alkyl)CO₂R₅,CO(C₁-C₆ alkyl)CO₂R₅, or CONH (C₁-C₆ alkyl)CO₂R₅.
 7. The compound ofclaim 1 wherein R₃ is (C₁-C₆ alkyl)CH(NHR₄)CO₂R₅, CO(C₁-C₆alkyl)CH(NHR₄)CO₂R₅, or CONH(C₁-C₆ alkyl)CH(NHR₄) CO₂R₅.
 8. The compoundof claim 1 wherein L is CO or NHCO.
 9. The compound of claim 1 wherein Lis a bond.
 10. The compound of claim 2 wherein Q is a group of theformula:

wherein R₂₀ is hydrogen or halogen.
 11. The compound of claim 6 whereinR₅ is hydrogen.
 12. A compound selected from the group consisting of:

wherein; X is F or H, m is zero to four, and n is one to four, or apharmaceutically-acceptable salt, solvate, or prodrug thereof.
 13. Acompound of claim 12 selected from the group consisting of:

wherein X is F or H, m is zero to four, and n is one to four, or apharmaceutically-acceptable salt, solvate, or prodrug thereof.
 14. Acompound of claim 12 selected from the group consisting of:

wherein X is F or H, or a pharmaceutically-acceptable salt, solvate, orprodrug thereof.
 15. A compound of claim 12 selected from the groupconsisting of:

wherein X is F or H, or a pharmaceutically-acceptable salt, solvate, orprodrug thereof.
 16. A compound of claim 12 selected from the groupconsisting of:

wherein X is F or H, m is zero to four, and n is one to four, or apharmaceutically-acceptable salt, solvate, or prodrug thereof.
 17. Acompound of claim 12 selected from the group consisting of:

wherein X is F or H, or a pharmaceutically-acceptable salt, solvate, orprodrug thereof.
 18. A compound having the formula:

wherein X is F or H and m is zero to four, or apharmaceutically-acceptable salt, solvate, or prodrug thereof.
 19. Acompound having the formula:

wherein X is F or H and n is one to four, or apharmaceutically-acceptable salt, solvate, or prodrug thereof.
 20. Acompound having the formula:

wherein X is F or H and n is one to four, or apharmaceutically-acceptable salt, solvate, or prodrug thereof.
 21. Acompound having the formula:

wherein X is F or H and m is zero to four, or apharmaceutically-acceptable salt, solvate, or prodrug thereof.
 22. Thecompound having the formula:

wherein X is F or H and n is one to four, or apharmaceutically-acceptable salt, solvate, or prodrug thereof.
 23. Thecompound having the formula:

or a pharmaceutically-acceptable salt, solvate, or prodrug thereof. 24.A composition for inhibiting the binding of fibrinogen to bloodplatelets in a mammal, comprising a compound of claim 1 and apharmaceutically-acceptable carrier.
 25. A composition for inhibitingthe aggregation of blood platelets in a mammal, comprising a compound ofclaim 1 and a pharmaceutically-acceptable carrier.
 26. A composition forpreventing or treating thrombosis in a mammal, comprising a compound ofclaim 1 and a pharmaceutically-acceptable carrier.
 27. A method forinhibiting the binding of fibrinogen to blood platelets in a mammal,which comprises administering to the mammal a composition of claim 24.28. A method for inhibiting the aggregation of blood platelets in amammal, which comprises administering to the mammal a composition ofclaim
 25. 29. A method for preventing or treating thrombosis in amammal, which comprises administering to the mammal a composition ofclaim
 26. 30. A composition for treating a mammal, to alleviate thepathological effects of atherosclerosis, arteriosclerosis, acutemyocardial infarction, chronic stable angina, unstable angina, transientischemic attacks and strokes, peripheral vascular disease, arterialthrombosis, preeclampsia, embolism, restenosis following angioplasty,carotid endarterectomy, and anastomosis of vascular grafts, comprising acompound of claim 1 and a pharmaceutically-acceptable carrier.
 31. Amethod of treating a mammal, to alleviate the pathological effects ofatherosclerosis, arteriosclerosis, acute myocardial infarction, chronicstable angina, unstable angina, transient ischemic attacks and strokes,peripheral vascular disease, arterial thrombosis, preeclampsia,embolism, restenosis following angioplasty, carotid endarterectomy, andanastomosis of vascular grafts; wherein the method comprisesadministering to said mammal at least one compound as claimed in ofclaim 1; wherein, said compound is administered to said mammal in anamount sufficient to inhibit binding of fibrinogen on glycoproteinIIb-IIIa sites in said mammal to thereby alleviate said effects.
 32. Apharmaceutical formulation containing as an active ingredient a compoundclaim 1, associated with one or more pharmaceutically-acceptablecarriers therefor.
 33. A composition for inhibiting the binding offibrinogen to blood platelets in a mammal, comprising a compound of anda pharmaceutically acceptable carrier.
 34. A composition for inhibitingthe aggregation of blood platelets in a mammal, comprising a compound ofand a pharmaceutically acceptable carrier.
 35. A composition forpreventing or treating thrombosis in a mammal, comprising a compound ofand a pharmaceutically acceptable carrier.
 36. A method for inhibitingthe binding of fibrinogen to blood platelets in a mammal, whichcomprises administering to the mammal a composition of claim
 33. 37. Amethod for inhibiting the aggregation of blood platelets in a mammalwhich comprises administering to the mammal a composition of claim 34.38. A method for preventing or treating thrombosis in a mammal, whichcomprises administering to the mammal a composition of claim
 35. 39. Acomposition for treating a mammal, to alleviate the pathological effectsof atherosclerosis, arteriosclerosis, acute myocardial infarction,chronic stable angina, unstable angina, transient ischemic attacks andstrokes, peripheral vascular disease, arterial thrombosis, preeclampsia,embolism, restenosis following angioplasty, carotid endarterectomy, andanastomosis of vascular grafts, comprising a compound of and apharmaceutically acceptable carrier.
 40. A method of treating a mammal,to alleviate the pathological effects of atherosclerosis,arteriosclerosis, acute myocardial infarction, chronic stable angina,unstable angina, transient ischemic attacks and strokes, peripheralvascular disease, arterial thrombosis, preeclampsia, embolism,restenosis following angioplasty, carotid endarterectomy, andanastomosis of vascular grafts, wherein the method comprisesadministering to said mammal at least one compound of; wherein saidcompound is administered to said mammal in an amount sufficient toinhibit binding of fibrinogen on glycoprotein IIb-IIIa sites in saidmammal to thereby alleviate said effects.
 41. A pharmaceuticalformulation containing as an active ingredient a compound of associatedwith one or more pharmaceutically acceptable carriers therefor.